Novel tilapia virus and uses thereof

ABSTRACT

The invention is directed to isolated Tilapia Lake Virus or TiLV, and isolated nucleic acids sequences and polypeptides thereof. The invention also relates to probes and primers, and to antibodies against antigens from TiLV, and use of these reagents for detecting the presence or absence of TiLV in an animal. The invention also relates to iRNAs which target nucleic acid sequences of TiLV. The invention is also related to immunogenic compositions, including antibodies and vaccines, for inducing an immune response against TiLV in an animal. The invention is also related to gene constructs and cells comprising TiLV and isolated nucleic acids sequences and polypeptides thereof for use in developing prophylactic and therapeutic agents.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application claims priority of U.S. Provisional ApplicationSer. No. 62/091,824, filed Dec. 15, 2014, which is hereby incorporatedby reference in its entirety.

FIELD OF THE INVENTION

The invention is in the field of viruses and is related to a new virusfound in tilapia, denoted Tilapia Lake Virus (TiLV). The inventionincludes isolated TiLV, and isolated nucleic acids sequences andpolypeptides thereof. The invention also relates to primers and probes.The invention also relates to antibodies against antigens from TiLV. Theinvention is related to methods for detecting the presence or absence ofTiLV in an animal using primers, probes, and antibodies. The inventionalso relates to iRNAs which target nucleic acid sequences of TiLV. Theinvention is also related to immunogenic compositions for inducing animmune response against TiLV in an animal.

BACKGROUND OF THE INVENTION

Tilapines are the second most important group of farmed fish worldwide,with production of 2.5 million tons annually (Food and AgricultureOrganization 2010), and they serve as a primary protein source in thedeveloping world. The Sea of Galilee (Kinneret Lake) in Israel is onemajor source of commercial fishing. In recent years, the catch fishquantities have been subjected to a persistent decline.

Although the lake hosts some 27 species of fish, encompassing members ofthe families Cichlidae, Cyprinidae, Mugillidae, and Claridae, only adecrease in the catch of tilapines (Cichlidae) was noticeable. The mainedible fish of the lake, Sarotherodon (Tilapia) galilaeus (St. Peter'sfish), saw annual yields decreased from 316 tons in 2005 to 51, 8, and45 tons in 2007, 2009, and 2010, respectively.

S. galilaeus contributes to the maintenance of the ecological balance ofthis lake. Hence, beyond its economic impact, the significant decline ofSt. Peter's fish populations, as well as the other lake tilapines (suchas Tilapia zilli [common tilapia], Oreochromis aureus [Jordan tilapia],and Tristamella simonis intermedia) represents a definite threat to theentire ecosystem.

This decrease in the lake tilapine in the Sea of Galilee was due to aserious emerging disease in wild populations of tilapine species,including S. galilaeus, T. zilli, O. aureus, and T. simonis intermedia,and as well as in the pond-raised hybrid tilapia O. niloticus×O. aureusin Israel. The association of disease outbreaks with seasonality (May toOctober, when the water has relatively high temperatures) furtherindicated the involvement of an infectious agent, since watertemperature affects the emergence of a wide range of parasitic,bacterial, and viral diseases of fish. Routine monitoring of knownparasites, bacterial, viral pathogens or toxins did not reveal anyabnormalities, and no causal agent was identified.

A similar outbreak of disease in tilapia was also found in Ecuador.

Thus, there was a need to identify the causal agent of this decline intilapia populations in the two different geographical locations, as wellas a need for tools and methods for detecting the presence of the causalagent within fish populations and protecting fish poulations from thecausal agent of this decline. This invention addresses these needs.

SUMMARY OF THE INVENTION

In certain aspects, the invention relates to the finding that the causalagent of the tilapines disease is a novel Tilapia Lake Virus (TiLV) RNAvirus. The wide distribution of the virus and the existence of differentclinical features of its associated disease make the results describedherein of significance to fish farming and wild-life preservation.

In certain aspects, the invention relates to diagnostic tools useful forscreening a sample for TiLV.

In certain aspects, the invention described herein relates to vaccinesfor the protection of fish from viral diseases. In certain aspects, theinvention relates to vaccine compositions for the prevention of TilapiaLake Virus (TiLV)-induced disease in fish, particularly tilapines. Incertain aspects invention relates to methods for using the vaccines toprotect tilapines from TiLV-induced disease.

The particular aspects, invention is related to isolated TiLV nucleicacid sequences (including cDNA sequences corresponding to sense oranti-sense TiLV RNA sequences) and peptides thereof. The invention isalso related to antibodies against antigens derived from TiLV. Theinvention is also related to iRNAs which target nucleic acid sequencesof TiLV. The invention is related to methods for detecting the presenceor absence of TiLV in an animal (e.g. in a fish). The invention is alsorelated to immunogenic compositions for inducing an immune responseagainst TiLV in an animal (e.g. in a fish).

In certain aspects, the invention relates to an isolated nucleic acidsequence having the sequence of any of SEQ ID NOs: 1-11, sequencescomplementary to any of SEQ ID NOs: 1-11, and fragments and variantsthereof. In certain embodiments, the nucleic acid is a DNA sequence,including cDNA. In certain embodiments, the nucleic acid is an RNAsequence.

In certain aspects, the invention relates to a synthetic nucleic acidcomprising an isolated (or non-isolated) nucleic acid having thesequence of SEQ ID NOs: 1-10, sequences complementary to any of SEQ IDNOs: 1-11, and variants thereof. These synthetic nucleic acids includeprimers and probes.

In certain aspects, the invention relates to a primer set fordetermining the presence or absence of TiLV in a biological sample,wherein the primer set comprises at least one synthetic nucleic acidsequence selected from the group consisting of the synthetic nucleicacids described herein.

In certain aspects, the invention relates to a method for determiningthe presence or absence of TiLV in a biological sample, the methodcomprising: a) contacting nucleic acid from a biological sample with atleast one primer which is a synthetic nucleic acid described herein, b)subjecting the nucleic acid and the primer to amplification conditions,and, c) determining the presence or absence of amplification product,wherein the presence of amplification product indicates the presence ofRNA associated with of TiLV the sample.

In certain aspects, the invention relates to oligonucleotide probes fordetermining the presence or absence of TiLV in a biological sample.

In certain aspects, the invention is directed to iRNA molecules whichtarget nucleic acids from TiLV, for example but not limited to any ofSEQ ID NOs: 1-11, and variants thereof, and silence a target gene.

In certain aspects, the invention relates to a method for reducing thelevels of a TiLV protein in an animal, viral mRNA in an animal or viraltiter in a cell of an animal, the method comprising administering to theanimal an iRNA described herein.

In certain aspects, the invention relates to an isolated polypeptideencoded by the nucleic acid of any of SEQ ID NOs: 1-11, nucleic acidswith sequences complementary to any of SEQ ID NOs: 1-11, and fragmentsand variants thereof.

In certain aspects, the invention relates to an isolated polypeptide ofSEQ ID NO: 12, and fragments and variants thereof.

In a further aspect, the invention provides a computer readable mediumhaving stored thereon: (i) a nucleic acid sequence selected from thegroup consisting of: a nucleic acid sequence of any of SEQ ID NOs: 1-11,a sequence substantially identical to a nucleic acid sequence of any ofSEQ ID NOs: 1-11, and a sequence variant of a nucleic acid sequence ofany of SEQ ID NOs; 1-11; or (ii) an amino acid sequence encoded by anucleic acid sequence selected from the group consisting of: a nucleicacid sequence of any of SEQ ID NOs: 1-11, a sequence substantiallyidentical to a nucleic acid sequence of any of SEQ ID NOs: 1-11, and asequence variant of a nucleic acid sequence of any of SEQ ID NOs; 1-11.

In certain aspects, the invention relates to an isolated antibody thatspecifically binds to a polypeptide of the invention (e.g. a polypeptideof SEQ ID NO: 12 or a polypeptide encoded by any or SEQ ID NOs: 1-11 orsequences complementary thereto or fragments or variants thereof).

In certain aspects, the invention relates to a method for determiningwhether or not a sample contains TiLV, the method comprising: a)contacting a biological sample with an antibody that specifically bindsto a polypeptide of any of an isolated (or non-isolated) polypeptide ofSEQ ID NO: 12 or fragments or variants thereof, or a polypeptide encodedby any of SEQ ID NOs: 1-11 or sequences complementary thereto orfragments or variants thereof, and b) determining whether or not theantibody binds to an antigen in the biological sample, wherein bindingindicates that the biological sample contains TiLV. In certainembodiments, the determining comprises use of a lateral flow assay orELISA.

In certain aspects, the invention relates to a method for determiningwhether or not a biological sample has been infected by TiLV, the methodcomprising: a) determining whether or not a biological sample containsantibodies that specifically bind to a polypeptide of any of an isolated(or non-isolated) polypeptide of SEQ ID NO: 12 or fragments or variantsthereof, or a polypeptide encoded by any or SEQ ID NOs: 1-11 orsequences complementary thereto or fragments or variants thereof.

In certain aspects, the present invention provides immunogeniccompositions capable of inducing an immune response against TiLVincluding TiLV of the invention comprising a nucleic acid of any of SEQID NOs: 1-11 or fragments or variants thereof, or comprising a cDNAsequence complementary to the sense or an anti-sense strand of any ofSEQ ID NOs: 1-11 or fragments or variants thereof, or comprising apolypeptide encoded by any of SEQ ID NOs: 1-11, or a cDNA sequencecomplementary to the sense or an anti-sense strand of any of SEQ ID NOs:1-11, or comprising a polypeptide comprising SEQ ID NO: 12 or fragmentsor variants thereof, or comprising a killed or attenuated TiLV.

In certain aspects, the invention relates to a method of inducing animmune response in an animal, the method comprising administering animmunogenic composition described herein.

In another aspect, the invention provides a method for preventing orreducing TiLV infection in an animal, the method comprisingadministering a TiLV immunogenic composition described herein.

In another aspect, the invention provides a method for preventing orreducing TiLV infection in an animal, the method comprisingadministering a TiLV antibody described herein.

In one embodiment, the method of administration is oral, immersion orinjection.

In another aspect, the invention provides for use of any of theimmunogenic compositions described herein in the manufacture of avaccine for the treatment or prevention of TiLV infection in an animal.

In certain aspects, the invention relates to an isolated viruscomprising at least 24 consecutive nucleotides from an isolated (ornon-isolated) nucleic acid having the sequence of any of SEQ ID NOs:1-11; an isolated (or non-isolated) nucleic acid complementary to thesequence of any of SEQ ID NOs: 1-11; an isolated (or non-isolated)nucleic acid having at least about 60% sequence identity to any of SEQID NOs: 1-11; or an isolated (or non-isolated) nucleic acid having atleast about 60% sequence identity to a nucleic acid complementary to thesequence of any of SEQ ID NOs: 1-11.

In certain aspects, the invention relates to an isolated viruscomprising at least 8 consecutive amino acids from the polypeptideencoded by an isolated (or non-isolated) nucleic acid having thesequence of any of SEQ ID NOs: 1-11; an isolated (or non-isolated)nucleic acid complementary to the sequence of any of SEQ ID NOs: 1-11;an isolated (or non-isolated) nucleic acid having at least about 60%sequence identity to any of SEQ ID NOs: 1-11; or an isolated (ornon-isolated) nucleic acid having at least about 60% sequence identityto a nucleic acid complementary to the sequence of any of SEQ ID NOs:1-11.

In certain aspects, the invention relates to an isolated cell comprisingan isolated (or non-isolated) nucleic acid having the sequence of any ofSEQ ID NOs: 1-11, sequences complementary to SEQ ID NOs: 1-11 andfragments and variant thereof described herein.

In certain aspects, the invention relates to a method for culturingcells comprising: a) infecting a cell with TiLV, or an isolated (ornon-isolated) nucleic acid having the sequence of any of SEQ ID NOs:1-11, sequences complementary to SEQ ID NOs: 1-11 and fragments andvariant thereof described herein, and b) culturing the cells.

In certain aspects, the invention relates to a method of testing a TiLVvaccine, comprising: a) contacting cells with a TiLV vaccine; b)contacting cells with TiLV; and c) measuring the number of cellsinfected with TiLV.

In certain aspects, the invention relates to a method of testing a TiLVdrug, comprising: a) contacting cells with a TiLV drug; b) contactingcells with TiLV; and c) measuring the number of cells infected withTiLV.

In certain aspects, the invention relates to a method of testing a TiLVdrug, comprising: a) contacting cells with TiLV; b) contacting cellswith a TiLV drug; and c) measuring the replication of TiLV.

In other aspects, the invention provides methods for identifying and/orgenerating anti-viral drugs. For example, in one aspect the inventionprovides methods for identifying drugs that bind to and/or inhibit thefunction of TiLV-encoded proteins of the invention, or that inhibit thereplication or pathogenicity of TiLV of the invention. Methods ofidentifying drugs that affect or inhibit a particular drug target, suchas high throughput drug screening methods, are well known in the art andcan readily be applied to the proteins and viruses of the presentinvention.

The present invention also provides for methods and tools for drugdesign, testing of agents, and tools for basic research into the causesand etiology of TiLV.

BRIEF DESCRIPTION OF THE FIGURES

For the purpose of illustrating the invention, there are depicted indrawings certain embodiments of the invention. However, the invention isnot limited to the precise arrangements and instrumentalities of theembodiments depicted in the drawings.

FIG. 1 shows characteristics of tilapia disease and pathologicalfindings in commercial hybrid tilapia (O. niloticus×O. aureus hybrid)(FIG. 1A and FIG. 1C to FIG. 1E) and in wild tilapia (S. galilaeus) fromthe Sea of Galilee (FIG. 1B and FIG. 1F to FIG. 1H). FIG. 1A is aphotograph showing tilapia disease outbreak in a commercial pondresulting in massive mortality (August 2013). FIG. 1B is a photograph ofa diseased tilapia demonstrating shrinkage of the eye and loss of ocularfunctioning (phthisis bulbi). FIG. 1C shows the gross pathology of skinincludes multifocal to coalescing dermal erosions and ulcers denoted byarrows. FIG. 1D shows hematoxylin and eosin stain of kidney andinterstitium. The arrows mark a dilated vein packed with large numbersof red blood cells (congestion). Hematoxylin and eosin (H&E) stain×10.FIG. 1E shows hematoxylin and eosin stain of brain and cortex. Thearrows mark dilated blood vessels packed with large numbers of red bloodcells within the leptomeninges and gray and white matter. H&E stain×10was used. FIG. 1F shows hematoxylin and eosin stain of brain and cortex.Perivascular cuffs of lymphocytes are encircled. H&E stain×40 was used.FIG. 1Ga shows shows hematoxylin and eosin stain of lens. Cataractouschanges are characterized by formation of eosinophilic sphericalstructures (morgagnian globules) accompanied by degeneration ofcrystalline fibers (encircled). H&E stain×10 was used. FIG. 1Gb showshows hematoxylin and eosin stain of control lens from healthy fish. H&Estain×10 was used. FIG. 1H shows shows hematoxylin and eosin stain ofeye and cornea. Loss of integrity of the overlying squamous epitheliumwith inflammatory infiltrate (arrowheads) and multiple capillarieswithin the stroma (neovascularization; encircled). The collagen fiberswithin the superficial stroma are smudged and are stained paleeosinophilic (corneal edema). H&E stain×10 was used.

FIG. 2 shows cytopathogenic effect or CPE induction in infected culturesand electron microscopy (EM) analyses. FIG. 2A shows images of E-11infected cells with CPE at day 5 postinoculation. Plaque formation andvacuolated cells are shown at the rims of the plaques. The centers oftwo plaques are marked with asterisks. FIG. 2B shows images of infectedprimary tilapia brain cells with CPE at day 10 postinoculation.Conversion of the typical elongated cells into swollen, rounded, andgranulated cells is marked with arrows. FIG. 2C and FIG. 2D are imagesof controls, mock-infected E-11 or primary tilapia brain, respectively.FIG. 2E and FIG. 2F are transmission EM of thin sections of infectedE-11 cells with electron-dense particles (diameter, 55 to 60 nm)aggregated and enclosed in the intracytoplasmic membrane (FIG. 2E,marked with an arrow) or within the cytoplasm (FIG. 2F). Scale bars, 200and 500 nm for FIG. 2E and FIG. 2F, respectively. FIG. 2G is an EM imageof negatively stained virions, pelleted from infected E11 culturesupernatants. Scale bar, 100 nm.

FIG. 3 shows PCR detection of TiLV. FIG. 3A show the detection of TiLVby PCR. Total RNA was extracted from brains of TiLV infected fish (lanes1 to 7) and a healthy fish (lane 10), as well as from E-11 and primarytilapia brain infected cell cultures (lanes 8 and 9, respectively), andwas used as a template for cDNA generation. A 250-bp fragment wasamplified with ME1 (SEQ ID NO: 23) and clone 7450/150R (SEQ ID NO: 16)primers. FIG. 3B shows that reverse transcription was required for PCRamplification of TiLV. Total RNA was extracted from the supernatant(lanes 1 and 2) or from cell extracts (lanes 3 and 4) of TiLV-infectedE-11 culture, or from naive E-11 culture (lanes 5 and 6). The sampleswere not treated with DNase, and reverse transcription was carried out(+) or not (−) prior to the PCR step. A “no RNA” negative control (lane7) was also included. A 491-bp fragment was amplified with the primersNested ext-1 (SEQ ID NO: 24) and Nested ext-2 (SEQ ID NO: 25). FIG. 3Cshows the results of nuclease sensitivity assays. Nuclease-protectednucleic acids were extracted from purified virions and were treated (+)or not (−) with reverse transcriptase and/or RNase I prior to PCRamplification with TiLV-specific primers (Nested ext-1 (SEQ ID NO: 24)and Nested ext-2 (SEQ ID NO: 25) resulting in amplified product, 491 bp)or SnRV-specific primers (Snakehead gag-pol fw (SEQ ID NO: 26) andSnakehead gag-pol rev (SEQ ID NO: 27), resulting in amplified product,284 bp). M is the DNA size marker.

FIG. 4 shows kinetics of TiLV-induced mortality. Fish were divided intogroups of 30 specific pathogen-free (SPF) fish. Groups 1 (solid diamond)and 2 (solid triangle) were infected by intraperitoneally (i.p.)injection or cohabitation, respectively. The control group (solidcircle) was composed of an identical number of fish inoculated with thesupernatants of naive E-11 cultures. Variability between the threeexperimental groups was determined by chi-square tests, in which a Pvalue of 0.05 was considered significant. Bars represent standarderrors.

FIG. 5 shows Northern Blot analysis of nucleic acids from diseasedtilapia in Israel and Ecuador. FIG. 5A shows Northern Blot analysis oftotal RNA extracted from diseased Ecuadorian tilapia liver and analyzedwith three mixes of probes representing segments 1, 4, 7, and 10 (Combo1); 3, 6, and 9 (Combo 2); or 2, 5, and 8 (Combo 3) to prevent signaloverlap from similar sized segments (three right-hand panels). InfluenzaA virus RNA hybridized with three probes representing HA, NA, and matrixproteins served as size references (left panel). FIG. 5B shows NorthernBlot analysis of total RNA extracted from culture cells infected withbrain derived TiLV from Israeli tilapia or from culture supernatant andanalyzed with the three mixes of probes. Lanes 5, 8, and 11 show resultsfrom extracts from infected culture cells (6 dpi); lanes 6, 9, and 12show results from extracts from infected culture supernatant; lanes 4,7, and 10 show results from extracts from non-infected culture cells.

FIG. 6 shows the results of conventional diagnostic PCR using primersdesigned to amplify segment 3 of the TiLV genome. The figure is an imageof an agarose gel electrophoresis of amplification products obtainedfrom nucleic acid extracts of TiLV-infected cell culture supernatant.Lane 1 and 12 are size markers. Lanes 2-10 are serial 2-fold dilutionsof nucleic acid extract. Lane 11 is a negative control of non-infectedcell culture supernatant.

FIG. 7 shows the results of real-time PCR. FIG. 7A is a calibrationcurve using quantitated plasmid standards representing TiLV segment 1target sequence, ranging from 1×10⁶ to 1×10¹ molecules per assay. FIG.7B is a calibration curve using quantitated plasmid standardsrepresenting tilapia beta-actin target sequence, ranging from 1×10⁶ to1×10¹ molecules per assay. FIG. 7C shows the detection of authentic TiLVin various tissue specimens of diseased tilapia from Israel usingprimers designed from and specific for TiLV nucleic acid sequences.

DETAILED DESCRIPTION OF THE INVENTION

Tilapines are important for the sustainability of ecological systems andserve as the second most important group of farmed fish worldwide. Theinvention described herein relates to the observation of significantmortality of wild and cultured tilapia both in Israel and Ecuador.

Reported herein is the isolation of a previously undescribed virus,designated Tilapia Lake Virus or TiLV, from spontaneously diseased fishand the induction of disease in tilapia by this agent. The incubation ofextracts from diseased, but not healthy, tilapines with cultures of fishcells (E-11 and primary tilapia brain cells) resulted in the appearanceof cytopathogenic effect or CPE in infected cultures. Moreover, theinoculation of supernatants, harvested from these cultures, into naivetilapines resulted in the appearance of disease. TiLV was reisolated incell cultures from experimentally infected fish, and the agent induced asimilar disease upon inoculation of new naive fish. Furthermore, anexperimentally induced disease was achieved with a purified TiLVobtained by endpoint dilutions. Of note, the signs of the naturallyoccurring disease (discoloration, skin patches, ocular alterations, andlethargy) were observed in the experimentally induced disease. The TiLVsequences were amplified from diseased fish and TiLV-infected cellcultures hut not from naive fish, mock-infected cultures, or culturesinfected by another agent (VNN).

Several lines of evidence indicated that this infectious agent is avirus. First, the agent went through 0.2 □m filters while retaining itsinfectivity, ruling out the possibility of infection by microorganismslarger than this filter size (such as bacteria and fungi). Second, theappearance of CPE after serial passages of the agent in cell culturesexcludes the possibility of a filterable toxin-induced CPE. Third,virion-like structures were visualized by EM in infected cells and inthe supernatants of cultures of these cells. Fourth, CPE activity wasdemonstrated for relatively dense fractions of sucrose gradients,similar to known assembled virions. Fifth, the encapsidated TiLV genomeis made of RNA, as evidenced by the fact that it was amplified by RT-PCRonly (and not by PCR) from samples of sick fish and from cell culturesthat were inoculated with extracts of such fish, as well as by the factthat this amplification was sensitive to initial digestion with RNase I.RNA genomes are only known to occur for viruses. EM analyses and thesensitivity of TiLV to organic solvents (ether or chloroform) furtherindicate that TiLV is an enveloped virus.

TiLV-induced disease in tilapines was achieved either by intraperitoneal(i.p.) injections or by cohabitation. The cohabitation mode oftransmission demonstrates the ability of TiLV to spread by thewaterborne route. It should be noted that in these experiments,relatively high mortality rates were observed for both the i.p. andwaterborne routes.

The existence of fish that survived the TiLV-induced disease stronglyindicated that an effective immune response against this pathogen can bemounted. This has important applications for future disease containmentstrategies. Besides the possibility of vaccine development, thedetermination of the susceptibility of different tilapia species to TiLVshould be considered a measure of disease containment. This notion isbased on the well-documented differences in disease resistance amongspecies of the same genus.

This work also provides molecular characterization of TiLV isolated fromdiseased fish in both Israel and Ecuador. Ten segments of the TiLVgenome were identified from virus in fish from both venues, resulting inSEQ ID NOs: 1-11. (SEQ ID NO: 1 is a shorter version of SEQ ID NO:9—both are segment 3 of the TiLV genome). Homology searches in the NCBIdatabase yielded a single hit for segment 1 of the genome (SEQ ID NO: 7)that indicated a very distant homology to orthomyxoviral RNA-dependentRNA polymerase. This further indicates that TiLV is a new emergingpathogen for tilapia. In light of the extensive commercial production oftilapia and the fact that tilapia serves as a primary protein source inthe developing world, it is highly important to diagnose this newpathogen. The amplification of TiLV sequences from diseased fish andTiLV-infected cultures, described in this work, provides the basis for aPCR-based diagnosis, allowing prompt screening, surveillance,epidemiological studies, and disease containment. Additionally, the TiLVsequences can be used for vaccines, i.e., immunological compositions,for inducing immune responses against TiLV in tilapia populations.

The TiLV identified herein is a novel RNA virus. Thus, cDNA nucleic acidsequences do not exist in nature. In other words, the nucleic acidsequences of SEQ ID NO: 1-11 are non-naturally occurring compositionsthat are markedly different in structure than naturally occurring TiLVRNA sequences. Additionally any embodiments of the invention such asprimers, probes, antibodies, immunogenic compositions and cells and celllines, comprising the nucleic acids of SEQ ID NOs: 1-11 would also notnaturally occur.

In certain aspects, the invention relates to the finding that theetiological agent of this disease is a novel RNA virus. In certainaspects, the invention also relates to the methods useful for isolatingand detecting the virus. As is described further herein, the virus,denominated Tilapia Lake Virus (TiLV), can be propagated in primarytilapia brain cells or in an E-11 cell line. In certain aspects, theinvention relates to the finding that the virus can induce a cytopathiceffect at 5 to 10 days postinfection. In certain aspects, the inventionrelates to electron microscopy findings showing the presence ofenveloped icosahedral particles of 55 to 75 nm. In certain aspects, theinvention relates to the finding that low-passage TiLV, injectedintraperitoneally in tilapia, induced a disease resembling the naturaldisease, which typically presents with lethargy, ocular alterations, andskin erosions, with greater than 80% mortality.

In certain aspects, the invention relates to the finding thathistological changes included congestion of the internal organs (kidneysand brain) with foci of gliosis and perivascular cuffing of lymphocytesin the brain cortex; ocular inflammation included endophthalmitis andcataractous changes of the lens. In certain aspects, the inventionrelates to the finding that the cohabitation of healthy and diseasedfish demonstrated that the disease is contagious and that mortalities(80 to 100%) occur within a few days. In certain aspects, the inventionrelates to the finding that fish surviving the initial mortality wereimmune to further TiLV infections, indicating the mounting of aprotective immune response.

In certain aspects, the invention relates to the finding that screeningcDNA libraries identified a TiLV-specific sequence, allowing the designof a PCR-based diagnostic test. In certain aspects, the inventionrelates to testing enabling the specific identification of TiLV intilapines. Such testing is helpful for controlling the spread of thisvirus worldwide.

Definitions

The terms used in this specification generally have their ordinarymeanings in the art, within the context of this invention and thespecific context where each term is used. Certain terms are discussedbelow, or elsewhere in the specification, to provide additional guidanceto the practitioner in describing the methods of the invention and howto use them. Moreover, it will be appreciated that the same thing can besaid in more than one way. Consequently, alternative language andsynonyms may be used for any one or more of the terms discussed herein,nor is any special significance to be placed upon whether or not a termis elaborated or discussed herein. Synonyms for certain terms areprovided. A recital of one or more synonyms does not exclude the use ofthe other synonyms. The use of examples anywhere in the specification,including examples of any terms discussed herein, is illustrative only,and in no way limits the scope and meaning of the invention or anyexemplified term. Likewise, the invention is not limited to itspreferred embodiments.

In accordance with the present invention, there may be numerous toolsand techniques within the skill of the art, such as those commonly usedin molecular immunology, cellular immunology, pharmacology, andmicrobiology. See, e.g., Sambrook et al. (2001) Molecular Cloning: ALaboratory Manual. 3rd ed. Cold Spring Harbor Laboratory Press: ColdSpring Harbor, N.Y.; Ausubel et al. eds. (2005) Current Protocols inMolecular Biology, John Wiley and Sons, Inc.: Hoboken, N.J.; Bonifacinoet al. eds. (2005) Current Protocols in Cell Biology, John Wiley andSons, inc.: Hoboken, N.J.; Coligan et al. eds. (2005) Current Protocolsin Immunology, John Wiley and Sons, Inc.: Hoboken, N.J.; Coico et al.eds. (2005) Current Protocols in Microbiology, John Wiley and Sons,Inc.: Hoboken, N.J.; Coligan et al. eds. (2005) Current Protocols inProtein Science, John Wiley and Sons, Inc.: Hoboken, N.J.; and Enna etal. eds. (2005) Current Protocols in Pharmacology, John Wiley and Sons,Inc.: Hoboken, N.J.

The singular forms “a,” “an,” and “the” include plural reference unlessthe context clearly dictates otherwise.

The term “about” is used herein to mean approximately, in the region of,roughly, or around. When the term “about” is used in conjunction with anumerical range, it modifies that range by extending the boundariesabove and below the numerical values set forth. In general, the term“about” is used herein to modify a numerical value above and below thestated value by a variance of 20%.

As used herein, “TiLV” refers to isolates of the Tilapia Lake Virusdescribed herein.

As used herein, a “TiLV gene” refers to any one of the genes or genesegments as described in SEQ ID Nos: 1-11, identified in the TiLVgenome.

As used herein, the term “animal” refers to a vertebrate, including, butnot limited to, fish, (e.g. tilapia).

“Substantially identical,” in the context of two nucleic acids orpolypeptides, refers to two or more sequences or subsequences that haveat least 98%, at least 99% or higher nucleotide or amino acid residueidentity, when compared and aligned for maximum correspondence, asmeasured using one of the following sequence comparison algorithms or byvisual inspection.

“Percent identity” in the context of two or more nucleic acids orpolypeptide sequences, refers to the percentage of nucleotides or aminoacids that two or more sequences or subsequences contain which are thesame. A specified percentage of nucleotides can be referred to such as:60% identity, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, or 99% or more identity over a specified region, whencompared and aligned for maximum correspondence over a comparisonwindow, or designated region as measured using one of the followingsequence comparison algorithms or by manual alignment and visualinspection.

A “comparison window”, as used herein, includes reference to a segmentof any one of the number of contiguous positions selected from the groupconsisting of from 20 to 600, usually about 50 to about 200, moreusually about 100 to about 150 in which a sequence can be compared to areference sequence of the same number of contiguous positions after thetwo sequences are optimally aligned. Methods of alignment of sequencesfor comparison are well-known in the art. Optimal alignment of sequencesfor comparison can be conducted, e.g., by the local homology algorithmof Smith and Waterman (1981) Adv. Appl. Math. 2: 482, by the homologyalignment algorithm of Needleman and Wunsch (1970) J. Mol. Biol. 48:443, by the search for similarity method of Pearson and Lipman (1988)Proc. Natl. Acad. Sci. U.S.A. 85: 2444, by computerized implementationsof these algorithms (FASTDB (Intelligenetics), BLAST (National Centerfor Biomedical Information), GAP, BESTFIT, FASTA, and TFASTA in theWisconsin Genetics Software Package, Genetics Computer Group, 575Science Dr., Madison, Wis.), or by manual alignment and visualinspection (see, e.g., Ausuhel).

In the context of nucleic acids base symbols can be used to represent aposition on a nucleic acid sequence that can have multiple possiblealternative. For example, “W” represents A or T; “S” represents C or G;“M” represents A or C; “K” represents G or T; “R” represents A or G; “Y”represents C or T; “B” represents C, G, or T; “D” represents A, G, or T;“H” represents A, C, or T; “V” represents A, C, or G.

It will be understood that, for the particular TiLV polypeptidesdescribed here, natural variations can exist between individual TiLVstrains. These variations may be demonstrated by (an) amino aciddifference(s) in the overall sequence or by deletions, substitutions,insertions, inversions or additions of (an) amino acid(s) in saidsequence Amino acid substitutions which do not essentially alterbiological and immunological activities, have been described, e.g. byNeurath et al in “The Proteins” Academic Press New York (1979). Aminoacid replacements between related amino 15 acids or replacements whichhave occurred frequently in evolution are, inter alia, Ser/Ala, Ser/Gly,Asp/Gly, Asp/Asn, Ile/Val (see Dayhof, M. D., Atlas of protein sequenceand structure, Nat. Biomed. Res. Found., Washington D.C., 1978, vol. 5,suppl. 3). Other amino acid substitutions include Asp/Glu, Thr/Ser,Ala/Gly, Ala/Thr, Ser/Asn, Ala/Val, Thr/Phe, Ala/Pro, Lys/Arg, Leu/Ile,Leu/Val and Ala/Glu. Based on this information, Lipman and Pearsondeveloped a method for rapid and sensitive protein comparison (Science(1985) 227:1435) and determining the functional similarity betweenhomologous proteins. Such amino acid substitutions of the exemplaryembodiments of this invention, as well as variations having deletionsand/or insertions are within the scope of the invention as long as theresulting proteins retain their immune reactivity. It is know thatpolypeptide sequences having one or more amino acid sequence variationsas compared to a reference polypeptide may still be useful forgenerating antibodies that bind the reference polypeptide.

Nucleic Acids and Uses Thereof

The present invention provides TiLV nucleic acid sequences. Thesenucleic acid sequences may be useful for, inter alia, expression ofTiLV-encoded proteins or fragments, variants, or derivatives thereof,generation of antibodies against TiLV proteins, generation of primersand probes for detecting TiLV and/or for diagnosing TiLV infection,generating immunogenic compositions against TiLV, and screening fordrugs effective against TiLV as described herein.

In certain aspects, the TiLV nucleic acid sequences are provided in SEQID NOs: 1-11, corresponding to TiLV genome segments (see Table 2).

In certain aspects, the invention is directed to a TiLV isolated nucleicacid sequence as provided in any of SEQ ID NOs: 1-11

In certain aspects, the invention is directed to an isolated nucleicacid complementary to any of SEQ ID NOs: 1-11.

In certain aspects, the invention relates to variants of TiLV nucleicacid sequence having greater that 60% similarity to the sequence of anyof SEQ ID NOs: 1-11.

In certain aspects, the invention is directed to isolated nucleic acidsequence variants of any of SEQ TD NOs: 1-11 and fragments thereof.Variants of any of SEQ ID NOs: 1-11 include, but are not limited to,nucleic acid sequences having at least from about 50% to about 55%identity to that of any of SEQ ID NOs: 1-11. Variants of any of SEQ IDNOs: 1-11 include, but are not limited to, nucleic acid sequences havingat least from about 55.1% to about 60% identity to that of any of SEQ IDNOs: 1-11. Variants of any of SEQ ID NOs: 1-11 include, but are notlimited to, nucleic acid sequences having at least from about 60.1% toabout 65% identity to that of any of SEQ ID NOs: 1-11. Variants of anyof SEQ ID NOs: 1-11 include, but are not limited to, nucleic acidsequences having at least from about 65.1% to about 70% identity to thatof any of SEQ ID NOs: 1-11. Variants of any of SEQ ID NOs: 1-11 include,but are not limited to, nucleic acid sequences having at least fromabout 70.1% to about 75% identity to that of any of SEQ ID NOs: 1-11.Variants of any of SEQ ID NOs: 1-11 include, but are not limited to,nucleic acid sequences having at least from about 75.1% to about 80%identity to that of any of SEQ ID NOs: 1-11. Variants of any of SEQ IDNOs: 1-11 include, but are not limited to, nucleic acid sequences havingat least from about 80.1% to about 85% identity to that of any of SEQ IDNOs: 1-11. Variants of any of SEQ ID NOs: 1-11 include, but are notlimited to, nucleic acid sequences having at least from about 85.1% toabout 90% identity to that of any of SEQ ID NOs: 1-11. Variants of anyof SEQ ID NOs: 1-11 include, but are not limited to, nucleic acidsequences having at least from about 90.1% to about 95% identity to thatof any of SEQ ID NOs: 1-11. Variants of any of SEQ ID NOs: 1-11 include,but are not limited to, nucleic acid sequences having about 95.1%, about95.5%, about 96%, about 96.5%, about 97%, about 97.5%, about 98%, about98.5%, about 99%, about 99.5% or about 99.9% identity to that of any ofSEQ ID NOs: 1-11. Programs and algorithms for sequence alignment andcomparison of % identity and/or homology between nucleic acid sequencesare well known in the art, and include BLAST, SIM alignment tool, and soforth.

In a further embodiment, the invention provides an isolated nucleic acidhaving a sequence substantially identical to a nucleic acid of any ofSEQ ID NOs: 1-11, or a fragment thereof. In a further embodiment, theinvention provides an isolated nucleic acid having a sequencesubstantially identical to a nucleic acid complementary to any of SEQ IDNOs: 1-11, or a fragment thereof.

In one embodiment, the invention is directed to an isolated nucleic acidsequence comprising from about 10 to about 50 consecutive nucleotidesfrom any one or SEQ ID NOs: 1-11 or a sequence complementary to any oneof SEQ ID NOs: 1-11 or a variant thereof. In one embodiment, theinvention is directed to an isolated nucleic acid sequence comprisingfrom about 10 to about 100 consecutive nucleotides from any one or SEQID NOs: 1-11 or a sequence complementary to any one of SEQ ID NOs: 1-11or a variant thereof. In one embodiment, the invention is directed to anisolated nucleic acid sequence comprising from about 10 to about 200consecutive nucleotides from any one or SEQ ID NOs: 1-11 or a sequencecomplementary to any one of SEQ ID NOs: 1-11 or a variant thereof. Inone embodiment, the invention is directed to an isolated nucleic acidsequence comprising from about 10 to about 300 consecutive nucleotidesfrom any one or SEQ ID NOs: 1-11 or a sequence complementary to any oneof SEQ ID NOs: 1-11 or a variant thereof. In one embodiment, theinvention is directed to an isolated nucleic acid sequence comprisingfrom about 10 to about 400 consecutive nucleotides from any one or SEQID NOs: 1-11 or a sequence complementary to any one of SEQ ID NOs: 1-11or a variant thereof. In one embodiment, the invention is directed to anisolated nucleic acid sequence comprising from about 10 to about 500consecutive nucleotides from any one or SEQ ID NOs: 1-11 or a sequencecomplementary to any one of SEQ ID NOs: 1-11 or a variant thereof. Inone embodiment, the invention is directed to an isolated nucleic acidsequence comprising from about 10 to about 600 consecutive nucleotidesfrom any one or SEQ ID NOs: 1-11 or a sequence complementary to any oneof SEQ ID NOs: 1-11 or a variant thereof. In one embodiment, theinvention is directed to an isolated nucleic acid sequence comprisingfrom about 10 to about 700 consecutive nucleotides from any one or SEQID NOs: 1-11 or a sequence complementary to any one of SEQ ID NOs: 1-11or a variant thereof. In one embodiment, the invention is directed to anisolated nucleic acid sequence comprising from about 10 to about 800consecutive nucleotides from any one or SEQ ID NOs: 1-11 or a sequencecomplementary to any one of SEQ ID NOs: 1-11 or a variant thereof. Inone embodiment, the invention is directed to an isolated nucleic acidsequence comprising from about 10 to about 900 or more consecutivenucleotides from any one or SEQ ID NOs: 1-11 or a sequence complementaryto any one of SEQ ID NOs: 1-11 or a variant thereof. In one embodiment,the invention is directed to an isolated nucleic acid sequencecomprising from about 10 to about 1000 or more consecutive nucleotidesfrom any one or SEQ ID NOs: 1-11 or a sequence complementary to any oneof SEQ ID NOs: 1-11 or a variant thereof. In one embodiment, theinvention is directed to an isolated nucleic acid sequence comprisingfrom about 10 to about 1200 or more consecutive nucleotides from any oneor SEQ ID NOs: 1-11 or a sequence complementary to any one of SEQ IDNOs: 1-11 or a variant thereof. In one embodiment, the invention isdirected to an isolated nucleic acid sequence comprising from about 10to about 1400 or more consecutive nucleotides from any one or SEQ IDNOs: 1-11 or a sequence complementary to any one of SEQ ID NOs: 1-11 ora variant thereof. In one embodiment, the invention is directed to anisolated nucleic acid sequence comprising from about 10 to about 1640 ormore consecutive nucleotides from any one or SEQ ID NOs: 1-11 or asequence complementary to any one of SEQ ID NOs: 1-11 or a variantthereof.

Encompassed by the invention are polynucleotide sequences that arecapable of hybridizing to the claimed polynucleotide sequences,including any of the nucleic acid sequences disclosed herein, andfragments thereof under various conditions of stringency Polynucleotideshomologous to the sequences illustrated in SEQ ID NOs: 1-11, can beidentified, e.g., by hybridization to each other under stringent orunder highly stringent conditions. The term “nucleic acid hybridization”refers to anti-parallel hydrogen bonding between two single-strandednucleic acids, in which A pairs with T (or U if an RNA nucleic acid) andC pairs with G. Nucleic acid molecules are “hybridizable” to each otherwhen at least one strand of one nucleic acid molecule can form hydrogenbonds with the complementary bases of another nucleic acid moleculeunder defined stringency conditions. The stringency of a hybridizationreflects the degree of sequence identity of the nucleic acids involved,such that the higher the stringency, the more similar are the twopolynucleotide strands. Stringency of hybridization is determined, e.g.,by (i) the temperature at which hybridization and/or washing isperformed, and (ii) the ionic strength and (iii) concentration ofdenaturants such as formamide of the hybridization and washingsolutions, as well as other parameters. Hybridization requires that thetwo strands contain substantially complementary sequences. Depending onthe stringency of hybridization, however, some degree of mismatches maybe tolerated. Under “low stringency” conditions, a greater percentage ofmismatches are tolerable (i.e., will not prevent formation of ananti-parallel hybrid). Hybridization conditions for various stringenciesare known in the art and are disclosed in detail in at least Sambrook etal.

In certain aspects, the invention relates to a synthetic nucleic acidcomprising the nucleotides of an isolated (or non-isolated) nucleic acidhaving the sequence of any of SEQ ID NOs: 1-11; an isolated (ornon-isolated) nucleic acid complementary to the sequence of any of SEQID NOs: 1-11; an isolated (or non-isolated) nucleic acid having at leastabout 60% sequence identity to any of SEQ ID NOs: 1-11; an isolated (ornon-isolated) nucleic acid having at least about 60% sequence identityto a nucleic acid complementary to the sequence of any of SEQ ID NOs:1-11; an isolated (or non-isolated) nucleic acid which comprises atleast 10 consecutive nucleotides of any of SEQ ID NOs: 1-11; an isolated(or non-isolated) nucleic acid which comprises at least 10 consecutivenucleotides of a nucleic acid complementary to the sequence of any ofSEQ ID NOs: 1-11; an isolated (or non-isolated) nucleic acid whichcomprises at least 10 consecutive nucleotides of a sequence having atleast about 60% identity to any of SEQ ID NOs: 1-11; or an isolated (ornon-isolated) nucleic acid which comprises at least 10 consecutivenucleotides of a sequence having at least about 60% identity to anucleic acid complementary to the sequence of any of SEQ ID NOs: 1-11.

In yet another aspect, the invention provides a synthetic nucleic acidwhich has a sequence consisting of from about 10 to about 30 consecutivenucleotides from a nucleic acid sequence selected from the group ofsequence consisting of SEQ ID NOs: 1-11.

In yet another aspect, the invention provides a synthetic nucleic acidwhich has a sequence consisting of from about 10 to about 30 consecutivenucleotides consisting of consecutive nucleotides having a sequencewhich is a variant of any of SEQ ID NOs: 1-11 having at least about95.1%, about 95.5%, about 96%, about 96.5%, about 97%, about 97.5%,about 98%, about 98.5%, about 99%, about 99.5% or about 99.9% identityto that of any of SEQ ID NOs: 1-11.

In yet another aspect, the invention provides a composition comprisingone or more nucleic acids which has a sequence consisting of from about10 to about 30 consecutive nucleotides from a nucleic acid sequenceselected from the group of sequence consisting of SEQ ID NOs: 1-11.

In yet another aspect, the invention provides a composition comprisingone or more nucleic acids which has a sequence consisting of from about10 to about 30 consecutive nucleotides consisting of consecutivenucleotides having a sequence which is a variant of any of SEQ ID NOs:1-11 having at least about 95.1%, about 95.5%, about 96%, about 96.5%,about 97%, about 97.5%, about 98%, about 98.5%, about 99%, about 99.5%or about 99.9% identity to that of any of SEQ ID NOs: 1-11.

In yet another aspect, the invention provides a synthetic nucleic acidwhich has a sequence consisting of from about 10 to about 30 consecutivenucleotides from a nucleic acid sequence which is complementary to anucleic acid sequence selected from the group of sequence consisting ofSEQ ID NOs: 1-11.

In yet another aspect, the invention provides a synthetic nucleic acidwhich has a sequence consisting of from about 10 to about 30 consecutivenucleotides which is complementary to a nucleic acid consisting ofconsecutive nucleotides having a sequence which is a variant of any ofSEQ ID NOs: 1-11 having at least about 95.1%, about 95.5%, about 96%,about 96.5%, about 97%, about 97.5%, about 98%, about 98.5%, about 99%,about 99.5% or about 99.9% identity to that of any of SEQ ID NOs: 1-11.

In yet another aspect, the invention provides a composition comprisingone or more nucleic acids which has a sequence consisting of from about10 to about 30 consecutive nucleotides from a nucleic acid sequencewhich is complementary to a nucleic acid sequence selected from thegroup of sequence consisting of SEQ ID NOs: 1-11.

In yet another aspect, the invention provides a composition comprisingone or more nucleic acids which has a sequence consisting of from about10 to about 30 consecutive nucleotides which is complementary to anucleic acid consisting of consecutive nucleotides having a sequencewhich is a variant of any of SEQ ID NOs: 1-11 having at least about95.1%, about 95.5%, about 96%, about 96.5%, about 97%, about 97.5%,about 98%, about 98.5%, about 99%, about 99.5% or about 99.9% identityto that of any of SEQ ID NOs: 1-11.

In other aspects the invention is directed to isolated nucleic acidsequences such as primers and probes, comprising nucleic acid sequencesof any of SEQ ID NOs: 1-11. Such primers and/or probes may be useful fordetecting the presence of TiLV of the invention, for example in samplesof bodily fluids such as blood, saliva, or urine from an animal, andthus may be useful in the diagnosis of TiLV infection. Such probes candetect polynucleotides of any of SEQ ID NOs: 1-11 in samples whichcomprise TiLV represented by any of SEQ ID NOs: 1-11. The isolatednucleic acids which can be used as primer and probes are of sufficientlength to allow hybridization with, i.e. formation of duplex with acorresponding target nucleic acid sequence, a nucleic acid sequences ofany of SEQ ID NOs: 1-11, or a fragment or variant thereof.

The isolated nucleic acid of the invention which can be used as primersand/or probes can comprise from about 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31,32, 33, 34, 35, 36, 37, 38, 39, 40 40, 45, 50, 55, 60, 65, 70, 75, 80,85, 90, 95 and 100 consecutive nucleotides from any of SEQ ID NOs: 1-11,or sequences complementary to any of SEQ ID NOs: 1-11 or variantsthereof. The invention is also directed to primer and/or probes whichcan be labeled by any suitable molecule and/or label known in the art,for example but not limited to fluorescent tags suitable for use in RealTime PCR amplification, for example TaqMan, cybergreen, TAMRA and/or FAMprobes; radiolabels, and so forth. Tn certain embodiments, theoligonucleotide primers and/or probe further comprises a detectablenon-isotopic label selected from the group consisting of: a fluorescentmolecule, a chemiluminescent molecule, an enzyme, a cofactor, an enzymesubstrate, and a hapten.

In another aspect, the invention provides an oligonucleotide probe whichcomprises from about 10 to about 50 nucleotides, wherein at least about10 contiguous nucleotides are at least 95% complementary to a nucleicacid target region within a TiLV nucleic acid sequence in any of SEQ IDNOs: 1-11, wherein the oligonucleotide probe hybridizes to the nucleicacid target region under moderate to highly stringent conditions to forma detectable nucleic acid target:oligonucleotide probe duplex. In oneembodiment, the oligonucleotide probe is at least about 95.5%, about96%, about 96.5%, about 97%, about 97.5%, about 98%, about 98.5%, about99%, about 99.5% or about 99.9% complementary to SEQ ID NOs: 1-11. Inanother embodiment the oligonucleotide probe consists essentially offrom about 10 to about 50 nucleotides.

Tn certain aspects, the invention is directed to primer sets comprisingisolated nucleic acids as described herein, which primer sets aresuitable for amplification of nucleic acids from samples which comprisesTiLV represented by any one of SEQ ID NOs: 1-11, or variants thereof.Primer sets can comprise any suitable combination of primers which wouldallow amplification of a target nucleic acid sequences in a sample whichcomprises TiLV represented any of SEQ ID NOs: 1-11, or variants thereof.Amplification can be performed by any suitable method known in the art,for example but not limited to PCR, RT-PCR, transcription mediatedamplification (TMA).

In certain aspects, the invention relates to a primer set fordetermining the presence or absence of TiLV in a biological sample,wherein the primer set comprises at least one synthetic nucleic acidsequence selected from the group consisting of the synthetic nucleicacid described herein.

In certain aspects, the invention provides a primer set for determiningthe presence or absence of TiLV in a biological sample, wherein theprimer set comprises at least one synthetic nucleic acid sequenceselected from the group consisting of: a synthetic nucleic acid whichhas a sequence consisting of from about 10 to about 30 consecutivenucleotides from a nucleic acid sequence selected from the group ofsequence consisting of SEQ ID NOs: 1-11 or variants thereof as describedherein; or a synthetic nucleic acid which has a sequence consisting offrom about 10 to about 30 consecutive nucleotides from a nucleic acidsequence which is complementary to a nucleic acid sequence selected fromthe group of sequence consisting of SEQ ID NOs: 1-11 or variants thereofas described herein.

Primer sets can be designed by those of skill in the art using thesequences of SEQ ID NOs: 1-11. Examples of primer pairs useful for thedetection methods using PCR are:

ME1 (SEQ ID NO: 23) and clone 7450/150R (SEQ ID NO: 16);

Nested ext-1 (SEQ ID NO: 24) and nested ext-2 (SEQ ID NO: 25);

NM-CLU7-SF1 (SEQ ID NO: 28) and NM-CLU7-SRI (SEQ ID NO: 29);

TiLV-CLU5-cF1 (SEQ ID NO: 30) and TiLV-CLU5-cR1 (SEQ ID NO: 31); and

CLU5-mRNA-qF1 (SEQ ID NO: 34) and CLU5-mRNA-qR1 (SEQ ID NO: 35),

with optional probe, CLU5-mRNA-Probe (SEQ ID NO: 36).

In certain aspects, the invention relates to a method for determiningthe presence or absence of TiLV in a biological sample, the methodcomprising: a) contacting nucleic acid from a biological sample with atleast one primer which is a synthetic nucleic acid of an isolated (ornon-isolated) nucleic acid having the sequence of any of SEQ ID NOs:1-11; an isolated (or non-isolated) nucleic acid complementary to thesequence of any of SEQ ID NOs: 1-11; an isolated (or non-isolated)nucleic acid having at least about 60% sequence identity to any of SEQID NOs: 1-11; an isolated (or non-isolated) nucleic acid having at leastabout 60% sequence identity to a nucleic acid complementary to thesequence of any of SEQ ID NOs: 1-11; an isolated (or non-isolated)nucleic acid which comprises at least 10 consecutive nucleotides of anyof SEQ ID NOs: 1-11: an isolated (or non-isolated) nucleic acid whichcomprises at least 10 consecutive nucleotides of a nucleic acidcomplementary to the sequence of any of SEQ ID NOs: 1-11; an isolated(or non-isolated) nucleic acid which comprises at least 10 consecutivenucleotides of a sequence having at least about 60% identity to any ofSEQ ID NOs: 1-11; or an isolated (or non-isolated) nucleic acid whichcomprises at least 10 consecutive nucleotides of a sequence having atleast about 60% identity to a nucleic acid complementary to the sequenceof any of SEQ ID NOs: 1-11, b) subjecting the nucleic acid and theprimer to amplification conditions, and, c) determining the presence orabsence of amplification product, wherein the presence of amplificationproduct indicates the presence of RNA associated with of TiLV thesample.

The invention also relates to a method for determining the presence orabsence of TiLV in a biological sample, the method comprising: a)contacting nucleic acid from a biological sample with a primer pairchosen from the group consisting of: ME1 (SEQ ID NO: 23) and clone7450/150R (SEQ ID NO: 16); Nested ext-1 (SEQ ID NO: 24) and nested ext-2(SEQ ID NO: 25); NM-CLU7-SF1 (SEQ ID NO: 28) and NM-CLU7-SRI (SEQ ID NO:29); TiLV-CLU5-cF1 (SEQ ID NO: 30) and TiLV-CLU5-cR1 (SEQ ID NO: 31);and CLU5-mRNA-qF1 (SEQ ID NO: 34) and CLU5-mRNA-qR1 (SEQ ID NO: 35), b)subjecting the nucleic acid and the primer pair to amplificationconditions, and, c) determining the presence or absence of amplificationproduct, wherein the presence of amplification product indicates thepresence of RNA associated with of TiLV the sample.

In other aspects, the invention is directed to expression constructs,for example but not limited, to plasmids and vectors which comprise thenucleic acid sequence of any of SEQ ID NOs: 1-11, complementarysequences thereof, fragments and variants thereof. Such expressionconstructs can be prepared by any suitable method known in the art. Suchexpression constructs are suitable for viral nucleic acid and/or proteinexpression and purification.

In certain aspects, the invention is directed to interfering RNA (iRNA)molecules which target nucleic acids from TiLV, for example but notlimited to any of SEQ ID NOs: 1-11, and variants thereof, and silence atarget gene.

In certain aspects, the invention provides an iRNA comprising a sensestrand having at least 15 contiguous nucleotides complementary to theanti-sense strand of a gene comprising a nucleic acid sequence selectedfrom the group of sequences consisting of SEQ ID NOs: 1-11.

In certain aspects, the invention provides an iRNA comprising ananti-sense strand having at least 15 contiguous nucleotidescomplementary to the sense strand of a gene comprising a nucleic acidsequence selected from the group of sequences consisting of SEQ ID NOs:1-11.

In certain aspects, the invention relates to iRNA comprising at least 15contiguous nucleotides of an isolated (or non-isolated) nucleic acidhaving the sequence of any of SEQ ID NOs: 1-11; an isolated (ornon-isolated) nucleic acid complementary to the sequence of any of SEQID NOs: 1-11; an isolated (or non-isolated) nucleic acid having at leastabout 60% sequence identity to any of SEQ ID NOs: 1-11; or an isolated(or non-isolated) nucleic acid having at least about 60% sequenceidentity to a nucleic acid complementary to the sequence of any of SEQID NOs: 1-11.

An “IRNA agent” (abbreviation for “interfering RNA agent”) as usedherein, is an RNA agent, which can down-regulate the expression of atarget gene, e.g. a TiLV gene. An iRNA agent may act by one or more of anumber of mechanisms, including post-transcriptional cleavage of atarget mRNA sometimes referred to in the art as RNAi, orpre-transcriptional or pre-translational mechanisms. An iRNA agent canbe a double stranded (ds) iRNA agent.

A “ds iRNA agent” (abbreviation for “double stranded iRNA agent”), asused herein, is an iRNA agent which includes more than one, and incertain embodiments two, strands in which interchain hybridization canform a region of duplex structure. A “strand” herein refers to acontiguous sequence of nucleotides (including non-naturally occurring ormodified nucleotides). The two or more strands may be, or each form apart of, separate molecules, or they may be covalently interconnected,e.g. by a linker, e.g. a polyethyleneglycol linker, to form but onemolecule. At least one strand can include a region which is sufficientlycomplementary to a target RNA. Such strand is termed the “antisensestrand”. A second strand comprised in the dsRNA agent which comprises aregion complementary to the antisense strand is termed the “sensestrand”. However, a ds iRNA agent can also be formed from a single RNAmolecule which is, at least partly; self-complementary, forming, e.g., ahairpin or panhandle structure, including a duplex region. In such case,the term “strand” refers to one of the regions of the RNA molecule thatis complementary to another region of the same RNA molecule.

iRNA agents as described herein, including ds iRNA agents and siRNAagents, can mediate silencing of a gene, e.g., by RNA degradation. Forconvenience, such RNA is also referred to herein as the RNA to besilenced. Such a gene is also referred to as a target gene. In certainembodiments, the RNA to be silenced is a gene product of a TiLV gene.

As used herein, the phrase “mediates RNAi” refers to the ability of anagent to silence, in a sequence specific manner, a target gene.“Silencing a target gene” means the process whereby a cell containingand/or secreting a certain product of the target gene when not incontact with the agent, will contain and/or secrete at least 10%, 20%,30%, 40%, 50%, 60%, 70%, 80%, or 90% less of such gene product whencontacted with the agent, as compared to a similar cell which has notbeen contacted with the agent. Such product of the target gene can, forexample, be a messenger RNA (mRNA), a protein, or a regulatory element.

In the anti-viral uses of the present invention, silencing of a targetgene can result in a reduction in “viral titer” in the cell or in theanimal, wherein “reduction in viral titer” refers to a decrease in thenumber of viable virus produced by a cell or found in an organismundergoing the silencing of a viral target gene. Reduction in thecellular amount of virus produced can lead to a decrease in the amountof measurable virus produced in the tissues of an animal undergoingtreatment and a reduction in the severity of the symptoms of the viralinfection. iRNA agents of the present invention are also referred to as“antiviral iRNA agents”.

In other aspects, the invention provides methods for reducing viraltiter in an animal, by administering to an animal, at least one iRNAwhich inhibits the expression of a TiLV gene.

In certain aspects, the invention provides a methods for reducing thelevels of a viral protein, viral mRNA, or viral titer in a cell in ananimal comprising: administering an iRNA agent to an animal, wherein theiRNA agent comprises a sense strand having at least 15 contiguousnucleotides complementary to a gene or genome segment from TiLVcomprising a nucleic acid sequence selected from the group of sequencesconsisting of SEQ ID NOs: 1-11 and an antisense strand having at least15 contiguous nucleotides complementary to the sense strand. In oneembodiment, the method further comprises co-administering a second iRNAagent to the animal, wherein the second iRNA agent comprises a sensestrand having at least 15 or more contiguous nucleotides complementaryto a second gene or genome segment from the TiLV comprising a nucleicacid sequence selected from the group of sequences consisting of SEQ IDNOs: 1-11 and an antisense strand having at least 15 contiguousnucleotides complementary to the sense strand.

In certain aspects, the invention provides a method for reducing thelevels of a viral protein from at least one gene of TiLV in a cell in ananimal comprising: administering an iRNA agent to an animal, wherein theiRNA agent comprises a sense strand having at least 15 contiguousnucleotides complementary to a gene from TiLV comprising a nucleic acidsequence selected from the group of sequences consisting of SEQ ID NOs:1-11 and an antisense strand having at least 15 contiguous nucleotidescomplementary to the sense strand.

Isolated Polypeptides and Uses Thereof

The invention is also directed to isolated polypeptides and variants andderivatives thereof. These polypeptides may be useful for multipleapplications, including, but not limited to, generation of antibodiesand generation of immunogenic compositions. For example, the inventionis directed to an isolated polypeptide of SEQ ID NO: 12. For example,the invention is also directed to any isolated polypeptide encoded bythe nucleic sequence acid of any of SEQ TD NO: 1-11, fragments andvariants thereof. A peptide of at least 8 amino acid residues in lengthcan be recognized by an antibody MacKenzie et al. (1984) Biochemistry23: 6544-6549). In certain embodiments, the invention is directed tofragments of the polypeptides described herein, which can, for example,be used to generate antibodies.

Thus, in certain aspects, the invention relates to an isolatedpolypeptide having at least about 80% sequence identity to thepolypeptide of SEQ ID NO: 12. In certain aspects, the invention relatesto an isolated polypeptide comprising at least 8 consecutive amino acidsof the polypeptide of SEQ ID NO: 12. In certain aspects, the inventionrelates to an isolated polypeptide comprising at least 8 amino acidshaving at least about 80% identity to the sequence of the polypeptide ofSEQ ID NO: 12.

In one aspect, the invention is directed to polypeptide variants of anisolated polypeptide of SEQ ID NO: 12. Variants the isolatedpolypeptides of SEQ ID NO: 12 include, but are not limited to,polypeptide sequences having at least from about 50% to about 55%identity to that of an isolated polypeptide of SEQ ID NO: 12. Variantsof an isolated polypeptide of SEQ ID NO: 12 include, but are not limitedto, polypeptide sequences having at least from about 55.1% to about 60%identity to that of an isolated polypeptide of SEQ ID NO: 12. Variantsof an isolated polypeptide of SEQ ID NO: 12 include, but are not limitedto, polypeptide sequences having at least from about 60.1% to about 65%identity to that of an isolated polypeptide of SEQ ID NO: 12. Variantsof an isolated polypeptide of SEQ ID NO: 12 include, but are not limitedto, polypeptide sequences having at least from about 65.1% to about 70%identity to that of an isolated polypeptide of SEQ ID NO: 12. Variantsof an isolated polypeptide of SEQ ID NO: 12 include, but are not limitedto, polypeptide having at least from about 70.1% to about 75% identityto that of an isolated polypeptide of SEQ ID NO: 12. Variants of anisolated polypeptide of SEQ ID NO: 12 include, but are not limited to,polypeptide sequences having at least from about 75.1% to about 80%identity to that of an isolated polypeptide of SEQ ID NO: 12. Variantsof an isolated polypeptide of SEQ ID NO: 12 include, but are not limitedto, polypeptide sequences having at least from about 80.1% to about 85%identity to that of an isolated polypeptide of SEQ ID NO: 12. Variantsof an isolated polypeptide of SEQ ID NO: 12 include, but are not limitedto, polypeptide sequences having at least from about 85.1% to about 90%identity to that of an isolated polypeptide of SEQ ID NO: 12. Variantsof an isolated polypeptide of SEQ ID NO: 12 include, but are not limitedto, polypeptide sequences having at least from about 90.1% to about 95%identity to that of an isolated polypeptide of SEQ ID NO: 12. Variantsof an isolated polypeptide of SEQ ID NO: 12 include, but are not limitedto, polypeptide sequences having at least from about 95.1%, about 95.5%,about 96%, about 96.5%, about 97%, about 97.5%, about 98%, about 98.5%,about 99%, about 99.5% or about 99.9% identity to that of an isolatedpolypeptide of SEQ ID NO: 12.

In one aspect, the invention is directed to polypeptide variants of anyisolated polypeptide encoded by the nucleic acid sequence of any of SEQID NOs: 1-11. Variants of any one of the isolated polypeptides encodedby the nucleic acid sequence acid of any of SEQ ID NOs: 1-11 include,but are not limited to, polypeptide sequences having at least from about50% to about 55% identity to that of any isolated polypeptide encoded bythe nucleic acid sequence of any of SEQ ID NOs: 1-11. Variants of anyisolated polypeptide encoded by the nucleic acid sequence of any of SEQID NOs: 1-11 include, but are not limited to, polypeptide sequenceshaving at least from about 55.1% to about 60% identity to that of anyisolated polypeptide encoded by the nucleic acid sequence of any of SEQID NOs: 1-11. Variants of any isolated polypeptide encoded by thenucleic acid sequence of any of SEQ ID NOs: 1-11 include, but are notlimited to, polypeptide sequences having at least from about 60.1% toabout 65% identity to that of any isolated polypeptide encoded by thenucleic acid sequence of any of SEQ ID NOs: 1-11. Variants of anyisolated polypeptide encoded by the nucleic acid sequence of any of SEQID NOs: 1-11 include, but are not limited to, polypeptide sequenceshaving at least from about 65.1% to about 70% identity to that of anyisolated polypeptide encoded by the nucleic acid sequence of any of SEQID NOs: 1-11. Variants of any isolated polypeptide encoded by thenucleic acid sequence of any of SEQ ID NOs: 1-11 include, but are notlimited to, polypeptide having at least from about 70.1% to about 75%identity to that of any isolated polypeptide encoded by the nucleic acidsequence of any of SEQ ID NOs: 1-11. Variants of any isolatedpolypeptide encoded by the nucleic acid sequence of any of SEQ ID NOs:1-11 include, but are not limited to, polypeptide sequences having atleast from about 75.1% to about 80% identity to that of any isolatedpolypeptide encoded by the nucleic acid sequence of any of SEQ ID NOs:1-11. Variants of any isolated polypeptide encoded by the nucleic acidsequence of any of SEQ ID NOs: 1-11 include, but are not limited to,polypeptide sequences having at least from about 80.1% to about 85%identity to that of any isolated polypeptide encoded by the nucleic acidsequence of any of SEQ ID NOs: 1-11. Variants of any isolatedpolypeptide encoded by the nucleic acid sequence of any of SEQ ID NOs:1-11 include, but are not limited to, polypeptide sequences having atleast from about 85.1% to about 90% identity to that of any isolatedpolypeptide encoded by the nucleic acid sequence of any of SEQ ID NOs:1-11. Variants of any isolated polypeptide encoded by the nucleic acidsequence of any of SEQ ID NOs: 1-11 include, but are not limited to,polypeptide sequences having at least from about 90.1% to about 95%identity to that of any isolated polypeptide encoded by the nucleic acidsequence of any of SEQ ID NOs: 1-11. Variants of any isolatedpolypeptide encoded by the nucleic acid sequence of any of SEQ ID NOs:1-11 include, but are not limited to, polypeptide sequences having atleast from about about 95.1%, about 95.5%, about 96%, about 96.5%, about97%, about 97.5%, about 98%, about 98.5%, about 99%, about 99.5% orabout 99.9% identity to that of any of SEQ ID NOs: 1-11.

The invention is also directed to a polypeptide encoded by a nucleicacid that is complementary to a nucleic acid sequence of any of SEQ IDNOs: 1-11 or a fragment or variant thereof.

The invention is directed to a polypeptide comprising from about 10 toabout 50 consecutive amino acids of SEQ ID NO: 12 or variants thereof.The invention is directed to a polypeptide comprising from about 10 toabout 100 consecutive amino acids of SEQ ID NO: 12 or variants thereof.The invention is directed to a polypeptide comprising from about 10 toabout 150 consecutive amino acids of SEQ ID NO: 12 or variants thereof.The invention is directed to a polypeptide comprising from about 10 toabout 200 consecutive amino acids of SEQ ID NO: 12 or variants thereof.The invention is directed to a polypeptide comprising from about 10 toabout 250 consecutive amino acids of SEQ ID NO: 12 or variants thereof.The invention is directed to a polypeptide comprising from about 10 toabout 300 consecutive amino acids of SEQ ID NO: 12 or variants thereof.The invention is directed to a polypeptide comprising from about 10 toabout 350 consecutive amino acids of SEQ ID NO: 12 or variants thereof.The invention is directed to a polypeptide comprising from about 10 toabout 400 consecutive amino acids of SEQ ID NO: 12 or variants thereof.The invention is directed to a polypeptide comprising from about 10 toabout 420 consecutive amino acids of SEQ ID NO: 12 or variants thereof.In certain embodiments, the invention is directed to isolated andpurified peptides.

In certain embodiments, the polypeptides of the present invention can besuitable for use as antigens to detect antibodies against SEQ ID NO: 12,and variants thereof. In other embodiments, the polypeptides of thepresent invention which comprise antigenic determinants can be used invarious immunoassays to identify animals exposed to and/or samples whichcomprise SEQ ID NO: 12, and variants thereof.

The invention is directed to a polypeptide comprising from about 10 toabout 50 consecutive amino acids from any isolated polypeptide encodedby the nucleic acid sequence of any of SEQ ID NOs: 1-11 or complementarysequences or variants thereof. The invention is directed to apolypeptide comprising from about 10 to about 100 consecutive aminoacids from any isolated polypeptide encoded by the nucleic acid sequenceof any of SEQ ID NOs: 1-11 or complementary sequences or variantsthereof. The invention is directed to a polypeptide comprising fromabout 10 to about 150 consecutive amino acids from any isolatedpolypeptide encoded by the nucleic acid sequence of any of SEQ ID NOs:1-11 or complementary sequences or variants thereof. The invention isdirected to a polypeptide comprising from about 10 to about 200consecutive amino acids from any isolated polypeptide encoded by thenucleic acid sequence of any of SEQ ID NOs: 1-11 or complementarysequences or variants thereof. The invention is directed to apolypeptide comprising from about 10 to about 250 consecutive aminoacids from any isolated polypeptide encoded by the nucleic acid sequenceof any of SEQ ID NOs: 1-11 or complementary sequences or variantsthereof. The invention is directed to a polypeptide comprising fromabout 10 to about 300 consecutive amino acids from any isolatedpolypeptide encoded by the nucleic acid sequence of any of SEQ ID NOs:1-11 or complementary sequences or variants thereof. The invention isdirected to a polypeptide comprising from about 10 to about 350consecutive amino acids from any isolated polypeptide encoded by thenucleic acid sequence of any of SEQ ID NOs: 1-11 or complementarysequences or variants thereof. The invention is directed to apolypeptide comprising from about 10 to about 400 consecutive aminoacids from any isolated polypeptide encoded by the nucleic acid sequenceof any of SEQ ID NOs: 1-11 or complementary sequences or variantsthereof. The invention is directed to a polypeptide comprising fromabout 10 to about 450 consecutive amino acids from any isolatedpolypeptide encoded by the nucleic acid sequence of any of SEQ ID NOs:1-11 or complementary sequences or variants thereof. The invention isdirected to a polypeptide comprising from about 10 to about 460consecutive amino acids from any isolated polypeptide encoded by thenucleic acid sequence of any of SEQ ID NOs: 1-11 or complementarysequences or variants thereof. The invention is directed to apolypeptide comprising from about 10 to about 470 consecutive aminoacids from any isolated polypeptide encoded by the nucleic acid sequenceacid of any of SEQ TD NOs: 1-11 or complementary sequences or variantsthereof. The invention is directed to a polypeptide comprising fromabout 10 to about 480 consecutive amino acids from any isolatedpolypeptide encoded by the nucleic acid sequence acid of any of SEQ IDNOs: 1-11 or complementary sequences or variants thereof. The inventionis directed to a polypeptide comprising from about 10 to about 490consecutive amino acids from any isolated polypeptide encoded by thenucleic acid sequence acid of any of SEQ ID NOs: 1-11 or complementarysequences or variants thereof. The invention is further directed apolypeptide comprising from about 10 to about 550 consecutive aminoacids or more from any isolated polypeptide encoded by the nucleic acidsequence acid of any of SEQ ID NOs: 1-11 or complementary sequences orvariants thereof. In certain embodiments, the invention is directed toisolated and purified peptides.

In certain embodiments, the polypeptides of the present invention can besuitable for use as antigens to detect antibodies against TiLVrepresented by any of SEQ ID NOs: 1-11, and variants thereof. In otherembodiments, the polypeptides of the present invention which compriseantigenic determinants can be used in various immunoassays to identifyanimals exposed to and/or samples which comprise TiLV represented by anyof SEQ ID NOs: 1-11, and variants thereof.

Antibodies, Methods of Making and Methods of Using

In another aspect, the invention is directed to an antibody whichspecifically binds to amino acids from the polypeptide of an isolatedpolypeptide of SEQ ID NO: 12, fragments and variants thereof, asdescribed herein. In one embodiment the antibody is purified. Theantibodies can be polyclonal or monoclonal. The antibodies can also bechimeric (i.e., a combination of sequences from more than one species,for example, a chimeric mouse-human immunoglobulin), humanized orfully-human. Species specific antibodies avoid certain of the problemsassociated with antibodies that possess variable and/or constant regionsfrom other species. The presence of such protein sequences from otherspecies can lead to the rapid clearance of the antibodies or can lead tothe generation of an immune response against the antibody by anantibody.

In another aspect, the invention is directed to an antibody whichspecifically binds to amino acids from the polypeptide of any isolatedpolypeptide encoded by the nucleic sequence acid of any of SEQ ID NOs:1-11, fragments and variants thereof, as described herein. In oneembodiment the antibody is purified. The antibodies can be polyclonal ormonoclonal. The antibodies can also be chimeric (i.e., a combination ofsequences from more than one species, for example, a chimericmouse-human immunoglobulin), humanized or fully-human. Species specificantibodies avoid certain of the problems associated with antibodies thatpossess variable and/or constant regions from other species. Thepresence of such protein sequences from other species can lead to therapid clearance of the antibodies or can lead to the generation of animmune response against the antibody by an antibody.

An antibody described in this application can include or be derived fromany mammal, such as but not limited to, a bird, a dog, a human, a mouse,a rabbit, a rat, a rodent, a primate, or any combination thereof andincludes isolated avian, human, primate, rodent, mammalian, chimeric,humanized and/or CDR-grafted or CDR-adapted antibodies, immunoglobulins,cleavage products and other portions and variants thereof.

Any method known in the art for producing antibodies can be used togenerate the antibodies described herein. Exemplary methods includeanimal inoculation, phage display, transgenic mouse technology andhybridoma technology.

The antibodies of the present invention can be used to modulate theactivity of any polypeptide encoded by the nucleic sequence acid of anyof SEQ ID NOs: 1-11, variants or fragments thereof. In certain aspects,the invention is directed to a method for treating an animal, the methodcomprising administering to the animal an antibody which specificallybinds to amino acids from the polypeptide of any polypeptide encoded bythe nucleic sequence acid of any of SEQ ID NOs: 1-11, fragments andvariants thereof, as described herein.

In certain embodiments, antibody binding to the polypeptide of anypolypeptide encoded by the nucleic sequence acid of any of SEQ ID NOs:1-11, fragments and variants thereof, as described herein, may interfereor inhibit the function of the polypeptide, thus providing a method toinhibit virus propagation and spreading. In other embodiments, theantibody binds to the polypeptide of any polypeptide encoded by thenucleic sequence acid of any of SEQ ID NOs: 1-11, fragments and variantsthereof, as described herein, and does not interfere or inhibit thefunction of the polypeptide.

In other embodiments, the antibodies of the invention can be used topurify a polypeptide of SEQ ID NO: 12, variants or fragments thereof asdescribed herein. In other embodiments, the antibodies of the inventioncan be used to identify expression and localization of the polypeptideof SEQ TD NO: 12, variants, fragments or domains thereof. Analysis ofexpression and localization of the polypeptide of SEQ ID NO: 12 can beuseful in determining potential role of the polypeptide of SEQ ID NO:12.

In other embodiments, the antibodies of the invention can be used topurify polypeptides of any polypeptide encoded by the nucleic acidsequence of any of SEQ ID NOs: 1-11, variants or fragments thereof asdescribed herein. In other embodiments, the antibodies of the inventioncan be used to identify expression and localization of the polypeptideof any polypeptide encoded by the nucleic acid sequence of any of SEQ IDNOs: 1-11, variants, fragments or domains thereof. Analysis ofexpression and localization of the polypeptide of any polypeptideencoded by the nucleic acid sequence of any of SEQ ID NOs: 1-11 can beuseful in determining potential role of the polypeptide of anypolypeptide encoded by the nucleic acid sequence of any of SEQ ID NOs:1-11 and fragments and variants thereof.

In other embodiments, the antibodies of the present invention can beused in various immunoassays to identify animals exposed to and/orsamples which comprise antigens from TiLV represented by SEQ ID NO: 12,and fragments and variants thereof, as described herein.

In other embodiments, the antibodies of the present invention can beused in various immunoassays to identify animals exposed to and/orsamples which comprise antigens from TiLV represented by any of SEQ IDNOs: 1-11, and fragments and variants thereof, as described herein.

Any suitable immunoassay which can lead to formation of antigen-antibodycomplex can also be used. Variations and different formats ofimmunoassays, for example but not limited to ELISA, lateral flow assaysfor detection of analytes in samples, immunoprecipitation, are known inthe art. In various embodiments, the antigen and/or the antibody can belabeled by any suitable label or method known in the art. For exampleenzymatic immunoassays may use solid supports, or immunoprecipitationImmunoassays which amplify the signal from the antigen-antibody immunecomplex can also be used with the methods described herein.

In certain aspects the invention provides methods for assaying a sampleto determine the presence or absence of a TiLV comprising SEQ ID NO: 12,and fragments and variants thereof, as described herein. In certainembodiments, methods for assaying a sample, include, but are not limitedto, methods which can detect the presence of nucleic acids, methodswhich can detect the presence of antigens, methods which can detect thepresence of antibodies against antigens from a polypeptide of SEQ ID NO:12, or a polypeptide of SEQ ID NO: 12, fragments and variants thereof,as described herein.

In certain aspects the invention provides methods for assaying a sampleto determine the presence or absence of a TiLV comprising any of SEQ IDNOs: 1-11, and fragments and variants thereof, as described herein. Incertain embodiments, methods for assaying a sample, include, but are notlimited to, methods which can detect the presence of nucleic acids,methods which can detect the presence of antigens, methods which candetect the presence of antibodies against antigens from polypeptidesencoded by any of SEQ ID NOs: 1-11, or any polypeptide encoded by thenucleic sequence acid of any of SEQ ID NOs: 1-11, fragments and variantsthereof, as described herein.

Kits

Also provided for are kits for practicing one or more of theabove-described methods.

The subject reagents and kits thereof may vary greatly. Reagents ofinterest include reagents specifically designed for use in determiningif an animal has TiLV.

One type of regent that is specifically tailored for the detection ofthe TiLV is at least one oligonucleotide primer specific SEQ ID NOs:1-11 to amplify nucleic acid obtained from a biological sample, and,optionally, at least one primer suitable to enable sequencing of theamplified nucleic acid and determination of the presence of themutation.

Examples of primers that can be included as reagents are:

ME1 (SEQ ID NO: 23) and clone 7450/150R (SEQ ID NO: 16);

Nested ext-1 (SEQ ID NO: 24) and nested ext-2 (SEQ ID NO: 25);

NM-CLU7-SF1 (SEQ ID NO: 28) and NM-CLU7-SRI (SEQ ID NO: 29);

TiLV-CLU5-cF1 (SEQ ID NO: 30) and TiLV-CLU5-cR1 (SEQ ID NO: 31); and

CLU5-mRNA-qF1 (SEQ ID NO: 34) and CLU5-mRNA-qR1 (SEQ ID NO: 35),

with optional probe, CLU5-mRNA-Probe (SEQ ID NO: 36).

A further type of reagent is one or more nucleic acid probes comprisingor complementary SEQ ID NOs: 1-11. In one embodiment, one or more probesare in an array formation. A variety of different array formats areknown in the art with a wide variety of different probe structures,substrate compositions, and attachment technologies. In someembodiments, the arrays include at least 2 nucleic acid probes, in amore preferred embodiment, at least 5 nucleic acid probes, in a morepreferred embodiment, at least 10 nucleic acid probes, in a morepreferred embodiment, at least 15 nucleic acid probes, in a morepreferred embodiment, at least 25 nucleic acid probes, and in a mostpreferred embodiment, at least 50 nucleic acid probes, said nucleic acidprobes comprising or complementary SEQ ID NOs: 1-11.

A further type of reagent is one or more antibodies as described hereinthat specifically binds to amino acids from the polypeptide of anisolated polypeptide of SEQ ID NO: 12, fragments and variants thereof,or amino acids from the polypeptide of any isolated polypeptide encodedby the nucleic sequence acid of any of SEQ ID NOs: 1-11, fragments andvariants thereof.

The kit of the invention may include the above-described primers,probes, arrays, and antibodies as well as additional reagents employedin the various methods, such as: labeling reagents; enzymes such asreverse transcriptase, DNA and RNA polymerases, and the like; variousbuffers, such as hybridization and washing buffers; signal generationand detection reagents; and reagents for isolation of nucleic acid froma sample. In addition, the kit may include instructions for practicingthe methods of the present invention.

The invention also covers systems for practicing one or more of theabove-described methods. The subject systems may vary greatly buttypically include at least one element to detect TiLV, i.e., one or morereagents described above for detection of TiLV, including primers,probes, arrays, antibodies, and additional reagents for practicing themethods of the invention.

Immunogenic Compositions, Methods of Making and Methods of Using

As used herein, the term immunogenic composition refers to a compositioncapable of inducing an immunogenic response in an animal or a cell. Asused herein, reference to an immunogenic composition can include avaccine.

In certain aspects, the present invention provides immunogeniccompositions capable of inducing an immune response against TiLVincluding TiLV of the invention comprising a nucleic acid of any of SEQID NOs: 1-11 or fragments or variants thereof, or comprising a cDNAsequence complementary to the sense or an anti-sense strand of any ofSEQ ID NOs: 1-11 or fragments or variants thereof, or comprising apolypeptide encoded by any of SEQ ID NOs: 1-11, or a cDNA sequencecomplementary to the sense or an anti-sense strand of any of SEQ ID NOs:1-11, or comprising a polypeptide comprising SEQ ID NO: 12 or fragmentsor variants thereof or comprising a killed or attenuated TiLV.

In certain aspects, the invention relates to an immunogenic compositioncomprising any TiLV nucleic acid or polypeptide described herein,including variants and fragments.

In one embodiment, the immunogenic compositions are capable ofameliorating the symptoms of a TiLV infection and/or of reducing theduration of a TiLV associated disease. In another embodiment, theimmunogenic compositions are capable of inducing protective immunityagainst TiLV associated disease. The immunogenic compositions of theinvention can be effective against the TiLV viruses disclosed herein,and may also be cross-reactive with, and effective against, multipledifferent clades and strains of TiLV, and against otherorthomyxoviruses.

In another embodiment, the invention provides a method of inducing animmune response in an animal, the method comprising administering a TiLVnucleic acid, a TiLV polypeptide or a TiLV immunogenic composition tothe animal. Methods for administering polypeptides to animals andmethods of generating immune responses in animals by administeringimmunogenic peptides in immmunogenically effective amounts are known inthe art.

The types of immunogenic composition encompassed by the inventioninclude, but are not limited to, attenuated live viral immunogeniccompositions, inactivated (killed) viral immunogenic compositions, andsubunit immunogenic compositions.

The TiLV viruses of the invention may be attenuated by removal ordisruption of those viral sequences whose products cause or contributeto the disease and symptoms associated with TiLV infection, and leavingintact those sequences required for viral replication. In this way anattenuated TiLV can be produced that replicates in animals, and inducesan immune response in animals, but which does not induce the deleteriousdisease and symptoms usually associated with TiLV infection. One ofskill in the art can determine which TiLV sequences can or should beremoved or disrupted, and which sequences should be left intact, inorder to generate an attenuated TiLV suitable for use as an immunogeniccomposition.

The novel TiLV of the invention may be also be inactivated, such as bychemical treatment, to “kill” the viruses such that they are no longercapable of replicating or causing disease in animals, but still inducean immune response in an animal. There are many suitable viralinactivation methods known in the art and one of skill in the art canreadily select a suitable method and produce an inactivated “killed”TiLV suitable for use as an immunogenic composition.

The immunogenic compositions of the invention may comprise subunitimmunogenic compositions. Subunit immunogenic compositions includenucleic acid immunogenic compositions such as DNA immunogeniccompositions, which contain nucleic acids that encode one or more viralproteins or subunits, or portions of those proteins or subunits. Whenusing such immunogenic compositions, the nucleic acid is administered tothe animal, and the immunogenic proteins or peptides encoded by thenucleic acid are expressed in the animal, such that an immune responseagainst the proteins or peptides is generated in the animal. Subunitimmunogenic compositions may also be proteinaceous immunogeniccompositions, which contain the viral proteins or subunits themselves,or portions of those proteins or subunits.

To make the nucleic acid and DNA immunogenic compositions of theinvention TiLV sequences disclosed herein may be incorporated into aplasmid or expression vector containing the nucleic acid that encodesthe viral protein or peptide. Any suitable plasmid or expression vectorcapable of driving expression of the protein or peptide in the animalmay be used. Such plasmids and expression vectors should include asuitable promoter for directing transcription of the nucleic acid. Thenucleic acid sequence(s) that encodes TiLV protein or peptide may alsobe incorporated into a suitable recombinant virus for administration tothe animal. Examples of suitable viruses include, but are not limitedto, vaccinia viruses, retroviruses, adenoviruses and adeno-associatedviruses. One of skill in the art could readily select a suitableplasmid, expression vector, or recombinant virus for delivery of TiLVnucleic acid sequences of the invention.

To produce the proteinaceous immunogenic compositions of the invention,TiLV nucleic acid sequences of the invention are delivered to culturedcells, for example by transfecting cultured cells with plasmids orexpression vectors containing TiLV nucleic acid sequences, or byinfecting cultured cells with recombinant viruses containing TiLVnucleic acid sequences. TiLV proteins or peptides may then be expressedin the cultured cells and purified. The purified proteins can then beincorporated into compositions suitable for administration to animals.Methods and techniques for expression and purification of recombinantproteins are well known in the art, and any such suitable methods may beused.

Subunit immunogenic compositions of the present invention may encode orcontain any of TiLV proteins or peptides described herein, or anyportions, fragments, derivatives or mutants thereof, that areimmunogenic in an animal. One of skill in the art can readily test theimmunogenicity of TiLV proteins and peptides described herein, and canselect suitable proteins or peptides to use in subunit immunogeniccompositions.

Production of the TiLV viruses and immunogenic compositions can also beperformed using a recombinant expression system that expresses TiLV, aTiLV protein, a fragment of a TiLV protein or a variant of a TiLV viralprotein. The expression system can comprise any suitable plasmid or alinear expression construct known in the art.

The TiLV viruses and immunogenic compositions described herein can beproduced in cells. Production of the TiLV viruses and immunogeniccompositions described herein may also be accomplished on any usefulmedia and permissive cell or tissues, which may be derived from fish orother animal cell lines. As used herein, a cell or a tissue can include,but is not limited to individual cells, tissues, organs, insect cells,fish cells, mammalian cells, hybridoma cells, primary cells, continuouscell lines, and/or genetically engineered cells, such as recombinantcells expressing a virus. For example, production of the TiLV virusesand immunogenic compositions can be in any cell type, including but notlimited to tilapia cells. Cell lines suitable for producing the TiLVviruses and immunogenic compositions described herein The cell culturesystem for producing the TiLV viruses and immunogenic compositionsdescribed herein can be a traditional adherent monolayer culture.Alternatively, suspension and microcarrier cell culture systems can alsobe utilized.

The immunogenic compositions described herein can comprise aninactivated or killed TiLV vaccine. Inactivated immunogenic compositioncan be made by methods well known in the art. For example, once TiLV ispropagated to high titers, TiLV antigenic mass could be obtained bymethods well known in the art. For example, the TiLV viral antigenicmass may be obtained by dilution, concentration, or extraction. All ofthese methods have been employed to obtain appropriate TiLV antigenicmass to produce immunogenic compositions. TiLV may be inactivated bytreatment with formalin (e.g. 0.1-10%), betapropriolactone (BPL) (e.g.0.01-10%), or with binary ethyleneimine (BET) (e.g. 1-10 mM), or usingother methods known to those skilled in the art.

In addition to killed TiLV production, various means of attenuation arealso possible and are well known and described in the art. Attenuationleading to modified live immunogenic compositions can also be used inconjunction with the compositions and methods described herein. Methodsof attenuation suitable for use with the viruses described hereininclude continuous passaging in cell culture, continuous passaging inanimals, various methods for generating genetic modifications andultraviolet or chemical mutagenesis.

Attenuation of TiLV may be achieved through cold-adaptation of an TiLVstrain. Cold-adapted TiLV virus strains may be produced by methods whichincludes passaging a wild-type TiLV virus, followed by selection forTiLV that grows at a reduced temperature. Cold-adapted TiLV can beproduced, for example, by sequentially passaging a wild-type TiLV inembryonated cells or chicken eggs at progressively lower temperatures,thereby selecting for certain members of TiLV mixture which stablyreplicate at the reduced temperature. A cold-adapted TiLV strain mayexhibit a temperature sensitive phenotype. A temperature sensitivecold-adapted TiLV replicates at reduced temperatures, but no longerreplicates at certain higher growth temperatures at which the wild-typeTiLV will replicate. A temperature at which a temperature sensitive TiLVwill grow is referred to herein as a “permissive” temperature for thattemperature sensitive TiLV, and a higher temperature at which thetemperature sensitive TiLV will not grow, but at which a correspondingwild-type TiLV will grow, is referred to herein as a “non-permissive”temperature for that temperature sensitive TiLV. A cold-adapted TiLV mayalso be produced through recombinant means. In this approach, one ormore specific mutations, associated with identified cold-adaptation,attenuation, temperature sensitivity, or dominant interferencephenotypes, can be identified and are introduced back into a wild-typeTiLV strain using a reverse genetics approach. Reverse genetics entailscan be performed using RNA polymerase complexes isolated fromTiLV-infected cells to transcribe artificial TiLV genome segmentscontaining the mutation(s), incorporating the synthesized RNA segment(s)into virus particles using a helper virus, and then selecting forviruses containing the desired changes.

Attenuation of a TiLV may be achieved by serial passaging of a wild-typeTiLV strain in cell culture. TiLV strain can be passaged in a variety ofcell systems until its ability to produce disease is lost whilst itsimmunogenic character is fully retained. Once inoculated into the host,TiLV may be capable of multiplication to some extent. For example,attenuated TiLV compositions can be prepared from cell line that hasbeen attenuated by serial passage including serial passage atsub-optimal temperatures to a state where it is no longer capable ofcausing disease, but still capable of eliciting a protective immuneresponse.

Suitable attenuated TiLV strains may also be obtained by serialpassaging to obtain an over-attenuated strain. The “over-attenuation”means that the number of passages for attenuation has been substantiallygreater than what is normally necessary for the removal ofpathogenicity. The attenuated TiLV retains its antigenicity after thesenumerous passages so that its immunogenic ability is not impaired. Suchstrains produce practically no symptoms or side effects whenadministered, and thus are safe and efficacious vaccines.

Methods of purification of inactivated virus are known in the art andmay include one or more of, for instance gradient centrifugation,ultracentrifugation, continuous-flow ultracentrifugation andchromatography, such as ion exchange chromatography, size exclusionchromatography, and liquid affinity chromatography. Additional methodsof purification include ultrafiltration and diafiltration.

Other examples of purification methods suitable for use in the inventioninclude polyethylene glycol or ammonium sulfate precipitation (secTrepanier et al. (1981) Journal of Virological Methods 3:201-711; Hagenet al. (1996) Biotechnology Progress 12:406-412; and Carlsson et al.(1994) Journal of Virological Methods 47:27-36) as well asultrafiltration and microfiltration (see Pay et al. (1985) Developmentsin Biological Standardization 60:171-174; Tsurumi et al. (1990) PolymerJournal 22:1085-1100; and Makino et al. (1994) Archives of Virology139:87-96).

Viruses can be purified using chromatography, such as ion exchange,chromatography. Chromatic purification allows for the production oflarge volumes of virus containing suspension. The viral product ofinterest can interact with the chromatic medium by a simpleadsorption/desorption mechanism, and large volumes of sample can beprocessed in a single load. Contaminants which do not have affinity forthe adsorbent pass through the column. The virus material can then beeluted in concentrated form.

Anion exchange resins that may be used include but are not limited toDEAE, and EMD TMAE. Cation exchange resins may comprise a sulfonicacid-modified surface. Viruses can be purified using ion exchangechromatography comprising a strong anion exchange resin (e.g. EMD TMAE)for the first step and EMD-SO₃ (cation exchange resin) for the secondstep. A metal-binding affinity chromatography step can optionally beincluded for further purification. (See, e.g., WO 97/06243).

A resin such as Fractogel EMD can also be used This syntheticmethacrylate based resin has long, linear polymer chains covalentlyattached and allows for a large amount of sterically accessible ligandsfor the binding of biomolecules without any steric hindrance.

Column-based liquid affinity chromatography is another purificationmethod that can be used invention. One example of a resin for use inpurification method is Matrex Cellufine Sulfate (MCS). MCS consists of arigid spherical (approximately 45-105.mu.m diameter) cellulose matrix of3,000 Dalton exclusion limit (its pore structure excludesmacromolecules), with a low concentration of sulfate ester functionalityon the 6-position of cellulose. As the functional ligand (sulfate ester)is relatively highly dispersed, it presents insufficient cationic chargedensity to allow for most soluble proteins to adsorb onto the beadsurface. Therefore the bulk of the protein found in typical virus pools(cell culture supernatants, e.g. pyrogens and most contaminatingproteins, as well as nucleic acids and endotoxins) are washed from thecolumn and a degree of purification of the bound virus is achieved.

Inactivated viruses may be further purified by gradient centrifugation,or density gradient centrifugation. For commercial scale operation acontinuous flow sucrose gradient centrifugation would be an option. Thismethod is widely used to purify antiviral immunogenic compositions andis known to one skilled in the art.

Additional purification methods which may be used to purify viruses ofthe invention include the use of a nucleic acid degrading agent, anucleic acid degrading enzyme, such as a nuclease having DNase and RNaseactivity, or an endonuclease, such as from Serratia marcescens, membraneabsorbers with anionic functional groups or additional chromatographicsteps with anionic functional groups (e.g. DEAE or TMAE). Anultrafiltration/diafiltration and final sterile filtration step couldalso be added to the purification method.

The purified viral preparation of the invention is substantially free ofcontaminating proteins derived from the cells or cell culture and cancomprises less than about 1000, 500, 250, 150, 100, or 50 pg cellularnucleic acid/.mu.g virus antigen, and less than about 1000, 500, 250,150, 100, or 50 pg cellular nucleic acid/dose. The purified viralpreparation can also comprises less than about 20 pg or less than about10 pg. Methods of measuring host cell nucleic acid levels in a viralsample are known in the art. Standardized methods approved orrecommended by regulatory authorities such as the WHO or the FDA can beused.

The immunogenic compositions of the invention comprise at least oneTiLV-derived immunogenic component, such as those described herein. Thecompositions may also comprise one or more additives including, but notlimited to, one or more pharmaceutically acceptable carriers, buffers,stabilizers, diluents, preservatives, solubilizers, liposomes orimmunomodulatory agents. Suitable immunomodulatory agents include, butare not limited to, adjuvants, cytokines, polynucleotide encodingcytokines, and agents that facilitate cellular uptake of TiLV-derivedimmunogenic component.

Immunogenic compositions for use in accordance with the presentinvention thus may be formulated in a conventional manner using one ormore physiologically acceptable carriers comprising excipients andauxiliaries which facilitate processing of the active compounds intopreparations which can be used to induce an immunogenic response. Theseimmunogenic compositions may be manufactured in a manner that is itselfknown, e.g. by means of conventional mixing, dissolving, granulating,dragee-making, levigating, emulsifying, encapsulating, entrapping orlyophilizing processes. Proper formulation is dependent upon the routeof administration chosen.

When a therapeutically effective amount of protein or other activeingredient of the present invention is administered by intravenous,cutaneous or subcutaneous injection, protein or other active ingredientof the present invention will be in the form of a pyrogen-free,parenterally acceptable aqueous solution. The preparation of suchparenterally acceptable protein or other active ingredient solutions,having due regard to pH, isotonicity, stability, and the like, is withinthe skill in the art. An isotonic vehicle such as Sodium ChlorideInjection, Ringer's Injection, Dextrose Injection, Dextrose and SodiumChloride Injection, Lactated Ringer's Injection, Hanks's solution,physiological saline buffer, or other vehicle as known in the art can beused to formulate the solution. The immunogenic composition of thepresent invention may also contain stabilizers, preservatives, buffers,antioxidants, or other additives known to those of skill in the art. Fortransmucosal administration, penetrants appropriate to the barrier to bepermeated are used in the formulation. Such penetrants are generallyknown in the art.

For oral administration, the compounds can be formulated readily bycombining the active compounds with immunogenic acceptable carriers wellknown in the art. Such carriers enable the compounds of the invention tobe formulated as powder, tablets, pills, dragees, capsules, liquids,gels, syrups, slurries, suspensions, solution, elixir, and the like, fororal ingestion by an animal to be treated. Immunogenic preparations fororal use can be obtained solid excipient, optionally grinding aresulting mixture, and processing the mixture of granules, after addingsuitable auxiliaries, if desired, to obtain tablets or dragee cores.Suitable excipients are, in particular, fillers such as sugars,including lactose, sucrose, mannitol, or sorbitol; cellulosepreparations such as, for example, maize starch, wheat starch, ricestarch, potato starch, gelatin, gum tragacanth, methyl cellulose,hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, and/orpolyvinylpyrrolidone (PVP). If desired, disintegrating agents may beadded, such as the cross-linked polyvinyl pyrrolidone, agar, or alginicacid or a salt thereof such as sodium alginate. Dragee cores areprovided with suitable coatings. For this purpose, concentrated sugarsolutions may be used, which may optionally contain gum arabic, talc,polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, and/ortitanium dioxide, lacquer solutions, and suitable organic solvents orsolvent mixtures. When administered in liquid form, a liquid carriersuch as water, petroleum, oils of animal or plant origin such as peanutoil, mineral oil, soybean oil, or sesame oil, or synthetic oils may beadded. The liquid form of the immunogenic composition may furthercontain physiological saline solution, dextrose or other saccharidesolution, or glycols such as ethylene glycol, propylene glycol orpolyethylene glycol. Dyestuffs or pigments may be added to the tabletsor dragee coatings for identification or to characterize differentcombinations of active compound doses.

Immunogenic preparations which can be used orally include push-fitcapsules made of gelatin, as well as soft, sealed capsules made ofgelatin and a plasticizer, such as glycerol or sorbitol. The push-fitcapsules can contain the active ingredients in admixture with fillersuch as lactose, binders such as starches, and/or lubricants such astalc or magnesium stearate and, optionally, stabilizers. In softcapsules, the active compounds may be dissolved or suspended in suitableliquids, such as fatty oils, liquid paraffin, or liquid polyethyleneglycols. In addition, stabilizers may be added. All formulations fororal administration should be in dosages suitable for suchadministration. For buccal administration, the compositions may take theform of tablets or lozenges formulated in conventional manner.

Capsules and cartridges may be formulated containing a powder mix of thecompound and a suitable powder base such as lactose or starch. Thecompounds may be formulated for parenteral administration by injection,e.g., by bolus injection or continuous infusion. Formulations forinjection may be presented in unit dosage form, e.g., in ampules or inmulti-dose containers, with an added preservative. The compositions maytake such forms as suspensions, solutions or emulsions in oily oraqueous vehicles, and may contain formulatory agents such as suspending,stabilizing and/or dispersing agents.

Immunogenic formulations for parenteral administration include aqueoussolutions of the active compounds in water-soluble form. Additionally,suspensions of the active compounds may be prepared as appropriate oilyinjection suspensions. Suitable lipophilic solvents or vehicles includefatty oils such as sesame oil, or synthetic fatty acid esters, such asethyl oleate or triglycerides, or liposomes. Aqueous injectionsuspensions may contain substances which increase the viscosity of thesuspension, such as sodium carboxymethyl cellulose, sorbitol, ordextran. Optionally, the suspension may also contain suitablestabilizers or agents which increase the solubility of the compounds toallow for the preparation of highly concentrated solutions.Alternatively, the active ingredient maybe in powder form forconstitution with a suitable vehicle, e.g., sterile pyrogen-free water,before use.

A carrier for hydrophobic compounds of the invention can be a co-solventsystem comprising benzyl alcohol, a nonpolar surfactant, awater-miscible organic polymer, and an aqueous phase. The co-solventsystem may be the VPD co-solvent system. VPD is a solution of 3% w/vbenzyl alcohol, 8% w/v of the nonpolar surfactant polysorbate 80, and65% w/v polyethylene glycol 300, made up to volume in absolute ethanol.The VPD co-solvent system (VPD:5W) consists of VPD diluted 1:1 with a 5%dextrose in water solution. This co-solvent system dissolves hydrophobiccompounds well, and itself produces low toxicity upon systemicadministration. Naturally, the proportions of a co-solvent system may bevaried considerably without destroying its solubility and toxicitycharacteristics. Furthermore, the identity of the co-solvent componentsmay be varied: for example, other low-toxicity nonpolar surfactants maybe used instead of polysorbate 80; the fraction size of polyethyleneglycol may be varied; other biocompatible polymers may replacepolyethylene glycol, e.g. polyvinyl pyrrolidone; and other sugars orpolysaccharides may substitute for dextrose.

Alternatively, other delivery systems for hydrophobic immunogeniccompounds may be employed. Liposomes and emulsions are well knownexamples of delivery vehicles or carriers for hydrophobic drugs.Liposomes include amphipathic agents such as lipids which exist inaggregated form as micelles, insoluble monolayers, liquid crystals, orlamellar layers in aqueous solution. Suitable lipids for liposomalformulation include, without limitation, monoglycerides, diglycerides,sulfatides, lysolecithins, phospholipids, saponin, bile acids, and thelike. Preparation of such liposomal formulations is within the level ofskill in the art.

Additionally, the compounds may be delivered using a sustained-releasesystem, such as semipermeable matrices of solid hydrophobic polymerscontaining the therapeutic agent. Various types of sustained-releasematerials have been established and are well known by those skilled inthe art. Sustained-release capsules may, depending on their chemicalnature, release the compounds for a few weeks up to over 100 days.Depending on the chemical nature and the biological stability of thetherapeutic reagent, additional strategies for protein or other activeingredient stabilization may be employed.

The immunogenic compositions also may comprise suitable solid or gelphase carriers or excipients. Examples of such carriers or excipientsinclude but are not limited to calcium carbonate, calcium phosphate,various sugars, starches, cellulose derivatives, gelatin, and polymerssuch as polyethylene glycols. Many of the active ingredients of theinvention may be provided as salts with immunogenicly compatible counterions. Such immunogenicly acceptable base addition salts are those saltswhich retain the biological effectiveness and properties of the freeacids and which are obtained by reaction with inorganic or organic basessuch as sodium hydroxide, magnesium hydroxide, ammonia, dialkylamine,dialkylamine, monoalkylamine, dibasic amino acids, sodium acetate,potassium benzoate, triethanol amine and the like.

The immunogenic composition of the invention may be in the form of acomplex of the protein(s) or other active ingredient of presentinvention along with protein or peptide antigens.

The immunogenic compositions and vaccines described herein can also bemultivalent immunogenic compositions that further comprise additionalpolypeptides or nucleic acid sequences encoding additional polypeptidesfrom other viruses.

The immunogenic compositions and vaccines described herein can also bemultivalent immunogenic compositions that further comprise additionalpolypeptide fragments or nucleic acid sequences encoding additionalpolypeptide fragments from other viruses.

The immunogenic compositions and vaccines described herein can also bemultivalent immunogenic compositions that further comprise additionalviruses (e.g. viruses that are either attenuated, killed or otherwisedeactivated) or nucleic acid sequences encoding additional viruses (e.g.viruses that are either attenuated, killed or otherwise deactivated).

The immunogenic compositions and vaccines described herein can alsocomprise fusions proteins, or nucleic acids encoding fusion proteinscomprising a TiLV polypeptide, or a fragment or a variant thereof, andat least one polypeptide, or a polypeptide fragment or variant fromanother virus.

Other viral polypeptides and nucleic acid sequence suitable for use inthe immunogenic compositions described herein are discussed in Tucker etal. (2000) “Assessment of DNA vaccine potential for juvenile Japaneseflounder Paralichthys olivaceus, through the introduction of reportergenes by particle bombardment and histopathology” Vaccine 19(7-8):801;Corbeil et al. (1999) “Evaluation of the protective immunogenicity ofthe N, P, M, NV, G proteins of infectious hematopoietic necrosis virusin rainbow trout Oncorhynchus mykiss using DNA vaccines” Dis. Aquat.Organ 39(1):29; Nusbaum et al. (2002) “Protective immunity induced byDNA vaccination of channel catfish with early and late transcripts ofthe channel catfish herpes virus (IHV-1)” Vet Immunol. Immunopathol.84:151; Clark et al. (1992) “Developmental expression of surface antigengenes in the parasitic cilate Ichtyophthirius multifiliis” Proc. Natl.Acad. Sci. 89(14):6363-6367; and Sato et al. (2000) “Expression of YAVproteins and vaccination against viral ascites among cultured juvenileyellowtail” Biosci. Biotechnol. Biochem. 64:1494. Numerous nucleic acidand amino acid sequences of fish pathogen antigens are known andaccessible through the Genbank databases and other sources.

Other additives that are useful in immunogenic composition and vaccineformulations are known and will be apparent to those of skill in theart.

In one aspect, vaccination of animals may be performed by directlyinjecting the TiLV polypeptides, fragments or variants thereof into theanimal to generate an immunogenic response. In certain embodiments, theTiLV polypeptide can be injected by themselves, or an immunogenic TiLVcompositions comprising other components, including for example,excipients, additives and adjuvants.

Vaccination may also be performed by direct vaccination with a DNAencoding a TiLV polypeptide. When using such vaccines, the nucleic acidis administered to the animal, and the immunogenic polypeptide(s)encoded by the nucleic acid are expressed in the animal, such that animmune response against the proteins or peptides is generated in theanimal. Subunit vaccines may also be proteinaceous vaccines, whichcontain the viral proteins or subunits themselves, or portions of thoseproteins or subunits. Any suitable plasmid or expression vector capableof driving expression of a polypeptide may be used. Plasmids andexpression vectors can include a promoter for directing transcription ofthe nucleic acid. The nucleic acid sequence encoding TiLV polypeptidesmay also be incorporated into a suitable recombinant virus foradministration to the animal. Examples of suitable viruses include, butare not limited to, vaccinia viruses, retroviruses, adenoviruses andadeno-associated viruses. One of skill in the art will be able to selecta suitable plasmid, expression vector, or recombinant virus for deliveryof the TiLV nucleic acid sequences of the invention. Direct vaccinationwith DNA encoding proteins has been successful for many differentproteins. (As reviewed in for example Donnelly et al. (1993) TheImmunologist 2: 20-26 (1993)).

Vaccination with the TiLV nucleic acids and polypeptides describedherein can also be performed using live recombinant carriers capable ofexpressing the polypeptides described herein. Live recombinant carriersare micro-organisms or viruses in which additional genetic information,e.g. a nucleic acid sequence encoding a TiLV polypeptide, or a fragmentthereof has been cloned. Fish infected with such live recombinantcarriers will produce an immunological response not only against theimmunogens of the carrier, but also against the TiLV polypeptide or TiLVpolypeptide fragment.

Alternatively, passive vaccination can be performed by raising TiLVantibodies in a first animal species (e.g. a rabbit), fromantibody-producing cell lines, or from in vitro techniques beforeadministering such antibodies (in purified or unpurified form) to secondanimal species. This type of passive vaccination can be used when thesecond animal is already infected with a TiLV. In some cases, passivevaccination can be useful where the infection in the second animalcannot, or has not had sufficient time to mount an immune response tothe infection.

Many methods for the vaccination of fish are known in the art. Forexample, vaccination with the TiLV nucleic acids and polypeptidesdescribed herein can be performed in fish by injection, immersion,dipping or through oral administration. The administration protocol canbe optimized in accordance with standard vaccination practice

For oral vaccination of tilapia, the TiLV nucleic acids, polypeptides orimmunogenic compositions described herein can be mixed with feed, coatedon the feed or be administered in an encapsulated form. In certainembodiments, vaccination may be performed by incubating live feed in aTiLV vaccine suspension prior to feeding an animal (e.g. a tilapia) suchthat ingestion of the live feed will cause the TiLV vaccine toaccumulate in the digestive tract of the animal undergoing vaccination.One skilled in the art will appreciate that these methods ofadministration may expose an antigen to potential breakdown ordenaturation and thus the skilled artisan will ensure that the method ofvaccination will be appropriate for a chosen antigen. In the case oforal vaccination, the vaccine may also be mixed with one or morecarriers. Carriers suitable for use in oral vaccination include bothmetabolizable and non-metabolizable substances.

Vaccination of tilapia can also be performed by immersion protocols.Skin and gill epithelia in fish have mucosal surfaces that contribute tothe recognition of pathogens by adsorbing antigens. Adsorption in turnresults in the activation of antibody producing cells as part of theimmune response. Thus in one embodiment, vaccination of fish with thepolypeptides described herein can be performed by immersing fish inwater containing a TiLV vaccine composition. At least two types ofimmersion vaccination can be used in conjunction with the polypeptidesdescribed herein. In dip vaccination, fish are immersed in watercomprising for a short period of time (e.g. about 30 seconds) in aconcentrated vaccine solution (e.g. 1 part vaccine, 9 parts water). Inbath vaccination, immersion occurs for longer periods of time (e.g.several hours) in water containing lower vaccine concentrations. Oneskilled in the art will readily be able to determine the dilution ofTiLV vaccine and the duration of immersion sufficient to induce animmune reaction in an immersion protocol.

Another method for vaccinating tilapia with the TiLV nucleic acids andpolypeptides described herein is by injection vaccination. In injectionvaccination, a vaccine is injected into the abdominal cavity of thefish. Although one skilled in the art can readily determine the properinjection point, a common site for needle insertion in tilapia is themidline of the abdomen, one pelvic fin length in front of the base ofthe pelvic fins. In certain embodiments, the TiLV nucleic acids,polypeptides or immunogenic compositions can be delivered into the bodycavity of the fish in an oil emulsion, or other adjuvants or additivesthat enhance and/or prolong immune responses. In addition tointraperitoneal injection, injection vaccination can also be performedby intramuscular injection. One skilled in the art will appreciate thatimproper handling and needle insertion can cause mortality of fish andthus light anesthesia may be used during the vaccination process toreduce stress and mechanical injury to the animals. The skilled artisanwill also appreciate that needles having the proper length and thicknesscan be important to ensure proper vaccination while avoiding secondarycomplications due to infection, inflammation or tissue damage.

The TiLV nucleic acids, polypeptides or immunogenic compositionsdescribed herein can also be delivered in the form of an aerosol spraypresentation from pressurized packs or a nebulizer, with the use of asuitable propellant, e.g., dichlorodifluoromethane,trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide orother suitable gas. In the case of a pressurized aerosol the dosage unitmay be determined by providing a valve to deliver a metered amount.Capsules and cartridges may be formulated containing a powder mix of thecompound and a suitable powder base such as lactose or starch.

The TiLV nucleic acids, polypeptides or immunogenic compositionsdescribed herein can be administered in any immunologically effectiveamount sufficient to trigger an immune response in an animal. In certaininstances, this amount can be between about 0.01 and about 1000micrograms of the TiLV nucleic acid, polypeptide or immunogeniccomposition per animal.

An “immunologically effective amount” of the compositions of theinvention may be administered to an animal or a human. As used herein,the term “immunologically effective amount” refers to an amount capableof inducing, or enhancing the induction of, the desired immune responsein an animal or a human. The desired response may include, inter alia,inducing an antibody or cell-mediated immune response, or both. Thedesired response may also be induction of an immune response sufficientto ameliorate the symptoms of a TiLV associated disease and/or provideprotective immunity in an animal or a human against subsequent challengewith a TiLV. An immunologically effective amount may be an amount thatinduces actual “protection” against TiLV associated diseases, meaningthe prevention of any of the symptoms or conditions resulting from TiLVassociated disease in animals or humans. An immunologically effectiveamount may also be an amount sufficient to delay the onset of symptomsand conditions associated with infection, reduce the degree or rate ofinfection, reduce in the severity of any disease or symptom resultingfrom infection, and reduce the viral load of an infected animal or ahuman.

One of skill in the art can readily determine what is an“immunologically effective amount” of the compositions of the inventionwithout performing any undue experimentation. An effective amount can bedetermined by conventional means, starting with a low dose of and thenincreasing the dosage while monitoring the immunological effects.Numerous factors can be taken into consideration when determining anoptimal amount to administer, including the size, age, and generalcondition of the animal, the presence of other drugs in the animal, thevirulence of the particular TiLV against which the animal is beingvaccinated, and the like. The actual dosage is can be chosen afterconsideration of the results from various animal studies.

The immunologically effective amount of the immunogenic composition maybe administered in a single dose, in divided doses, or using a“prime-boost” regimen. The compositions may be administered by anysuitable route, including, but not limited to parenteral, intradermal,transdermal, subcutaneous, intramuscular, intravenous, intraperitoneal,intranasal, oral, or intraocular routes, or by a combination of routes.The compositions may also be administered using a “gun” device whichfires particles, such as gold particles, onto which compositions of thepresent invention have been coated, into the skin of an animal. Theskilled artisan will be able to formulate the immunogenic compositionaccording to the route chosen.

Dose sizes of the immunogenic compositions described herein can be inthe range of about 0.1 to 2.0 ml depending on the route ofadministration, but dose sizes are not limited to this range. Forinactivated TiLV compositions can contain suitable TCID₅₀ levels ofvirus prior to inactivation. The antigen content in TiLV preparation canhave, but is not limited to, a titer of between 10 to 10,000 units/ml asthe amount administered per dose. One of skill in the art will readilybe capable of determining a suitable antigen content for the immunogeniccompositions described herein.

For immunogenic compositions containing modified live TiLV or attenuatedTiLV, a therapeutically effective dose can be determined by one of skillin the art. For immunogenic compositions containing TiLV subunitantigens, a therapeutically effective dose can be determined by one ofskill in the art. While the amounts and concentrations of adjuvants andadditives useful in the context of the present invention can readily bedetermined by the skilled artisan.

Cells Comprising TiLV and Uses Thereof

TiLV can be used to infect cells. Cells may be cultured in any usefulmedia and any permissive cell or tissues, which may be, or may bederived from fish cell, including, but not limited to tilapia cells,CHSE-214 Oncorhynchus tshawytscha cells, BF-2 Lepomis macrocturus cells;BB Ictalurus nebulosus cells, EPC Cyprinus carpio cells, KF-1 Cyprinuscarpio cells, RTG-2 Salmo gairdneri cells, FHM Pimephales promelascells, and E-11 Ophicephalus striatus cells. As used herein, a cell or atissue can include, but is not limited to individual cells, tissues,organs, insect cells, rodent cells, avian cells, mammalian cells,hybridoma cells, primary cells, continuous cell lines, and/orgenetically engineered cells. Cell lines suitable for propagating,growing, or harboring TiLV nucleic acid sequence or for expressing apolypeptide produced by the TiLV nucleic acid sequence include, tilapiacells, CHSE-214 Oncorhynchus tshawytscha cells, BF-2 Lepomis macrocturuscells; BB Ictalurus nebulosus cells, EPC Cyprinus carpio cells, KF-1Cyprinus carpio cells, RTG-2 Salmo gairdneri cells, FHM Pimephalespromelas cells, and E-11 Ophicephalus striatus cells as well as non-fishcells but are not limited to dog kidney cells, BSC-1 cells, LLC-MKcells, CV-1 cells, CHO cells, COS cells, murine cells, human cells, HeLacells, 293 cells, VERO cells, MDBK cells, MDCK cells, MDOK cells, CRFKcells, RAF cells, TCMK cells, LLC-PK cells, PK15 cells, WI-38 cells,MRC-5 cells, T-FLY cells, BHK cells, SP2/0 cells, NS0, PerC6 (humanretina cells), chicken embryo cells or derivatives, embryonated eggcells, embryonated chicken eggs or derivatives thereof.

Cell culture media formulations to suitable for culturing cells infectedwith TiLV viruses described herein include, but are not limited to,Modified Eagle's media MEM, minimum essential media MEM, Dulbecco'smodified Eagle's media D-MEM, D-MEM-F12 media, William's E media, RPMImedia, HyClone cell culture medium (HyClone, Logan, Utah); andserum-free basal epithelial medium (CellnTech), and analogues andderivative thereof. These can also be specialty cell cultivation andvirus growth media as VP-SFM, OptiPro™. SFM, AIM V. R media, HyQ SFM4MegaVir, EX-CELL Vero SFM, EPISERF, ProVero, any 293 or CHO media andanalogues and derivatives thereof. The culture media described hereincan be supplemented by any additive known from prior art that isapplicable for cell and virus cultivation as for example animal sera andfractions or analogues thereof, amino acids, growth factors, hormones,buffers, trace elements, trypsin, sodium pyruvate, vitamins, L-glutamineand biological buffers. One medium is OptiPRO SEM supplemented withL-glutamine and trypsin. In certain embodiments, the cell culture mediacan be supplemented with 0.1 to 10 units of trypsin. Alternatively,plant derived equivalents of trypsin (e.g. Accutase) ranging from 2-100units can also be used in cell culture. Cell culture media can be usedin the absence or presence of animal-derived components. An example ofsupplementation with an animal-derived component is gamma-irradiatedserum ranging from 0.5-10% final concentration.

An expression vector can be introduced into cells in order to produceproteins (for example, SEQ ID NO: 12 and fragments and variantsdescribed thereof herein) encoded by nucleotide sequences of theinvention (for example any of SEQ ID NOs: 1-11 and fragments andvariants thereof described herein). Cells can harbor an expressionvector via introducing the expression vector into an appropriate hostcell via methods known in the art.

An expression vector can be introduced into cells in order to produceproteins encoded by nucleotide sequences of the invention (for exampleany of SEQ ID NOs: 1-11 or a sequence complementary to any of SEQ IDNOs: 1-11 and fragments and variants thereof described herein). Cellscan harbor an expression vector via introducing the expression vectorinto an appropriate host cell via methods known in the art.

A eukaryotic expression vector can be used to transfect cells in orderto produce proteins (for example, SEQ ID NO: 12 or proteins encoded bySEQ ID NOs: 1-11) and fragments and variants described herein encoded bynucleotide sequences of the vector.

An exogenous nucleic acid (for example any of SEQ ID NOs: 1-11, a cDNAof any of SEQ ID NOs: 1-11 or a cDNA complementary to any of SEQ ID NOs:1-11, fragments, or variants thereof described herein) can be introducedinto a cell via a variety of techniques known in the art.

A eukaryotic expression vector can be used to transfect cells in orderto produce proteins encoded by nucleotide sequences (for example any ofSEQ ID NOs: 1-11, a cDNA of any of SEQ ID NOs: 1-11 or a cDNAcomplementary to any of SEQ ID NOs: 1-11, fragments, or variants thereofdescribed herein). Mammalian cells can harbor an expression vector viaintroducing the expression vector into an appropriate host cell viamethods known in the art.

An exogenous nucleic acid can be introduced into a cell via a variety oftechniques known in the art, such as lipofection, microinjection,calcium phosphate or calcium chloride precipitation,DEAE-dextrin-mediated transfection, or electroporation. Other methodsused to transfect cells can also include calcium phosphateprecipitation, modified calcium phosphate precipitation, polybreneprecipitation, microinjection liposome fusion, and receptor-mediatedgene delivery. The expression vectors can contain coding sequences, orportions thereof, encoding the proteins for expression and production.Expression vectors containing sequences encoding the produced proteinsand polypeptides, as well as the appropriate transcriptional andtranslational control elements, can be generated using methods wellknown to and practiced by those skilled in the art. These methodsinclude synthetic techniques, in vitro recombinant DNA techniques, andin vivo genetic recombination which are described in Sambrook et al.,and Ausubel et al.

Cells to be infected with TiLV or nucleic acids thereof (for example anyof SEQ ID NOs: 1-11, a cDNA of any of SEQ ID NOs: 1-11 or a cDNAcomplementary to any of SEQ ID NOs: 1-11, fragments, or variantsthereof) can be primary and secondary cells, which can be obtained fromvarious tissues and include cell types which can be maintained andpropagated in culture.

Cells maintained in culture can be passaged by their transfer from aprevious culture to a culture with fresh medium. In one embodiment,induced epithelial cells are stably maintained in cell culture for atleast 3 passages, at least 4 passages, at least 5 passages, at least 6passages, at least 7 passages, at least 8 passages, at least 9 passages,at least 10 passages, at least 11 passages, at least 12 passages, atleast 13 passages, at least 14 passages, at least 15 passages, at least20 passages, at least 25 passages, or at least 30 passages.

In one embodiment, cells that have been infected with TiLV or containnucleic acids thereof (for example any of SEQ ID NOs: 1-11, a cDNA ofany of SEQ ID NOs: 1-11 or a cDNA complementary to any of SEQ ID NOs:1-11, fragments, or variants thereof described herein) can express avariety of markers that distinguish them from uninfected cells.Expression of markers can be evaluated by a variety of methods known inthe art. The presence of markers can be determined at the DNA, RNA orpolypeptide level.

In one embodiment, the method can comprise detecting the presence of amarker gene polypeptide expression. Polypeptide expression includes thepresence of a marker gene polypeptide sequence, or the presence of anelevated quantity of marker gene polypeptide as compared tonon-epithelial cells. These can be detected by various techniques knownin the art, including by sequencing and/or binding to specific ligands(such as antibodies). For example, polypeptide expression maybeevaluated by methods including, but not limited to, immunostaining, FACSanalysis, or Western blot. These methods are well known in the art

In another embodiment, the method can comprise detecting the presence ofnucleic acids (for example any of SEQ ID NOs: 1-11, a cDNA of any of SEQID NOs: 1-11 or a cDNA complementary to any of SEQ ID NOs: 1-11,fragments, or variants thereof). RNA expression includes the presence ofan RNA sequence, the presence of an RNA splicing or processing, or thepresence of a quantity of RNA. These can be detected by varioustechniques known in the art, including by sequencing all or part of themarker gene RNA, or by selective hybridization or selectiveamplification of all or part of the RNA. In one embodiment, in situhybridization can be used to detect TiLV nucleic acids.

The resulting transformed cells can be used for basic research as wellas testing for therapeutic and prophylactic agents. Specifically, forthe latter, the host cells can be incubated and/or contacted with apotential therapeutic or prophylactic agent. The resulting expression ofthe gene construct can be detected and compared to the expression of thegene construct in the cell before contact with the agent.

These gene constructs as well as the host cells transformed with thesegene constructs can also be the basis for transgenic animals for testingboth as research tools and for therapeutic and prophylactic agents. Suchanimals would include but are not limited to, nude mice and fish.Phenotypes can be correlated to the genes and looked at in order todetermine the genes effect on the animals as well as the change inphenotype after administration or contact with a potential therapeuticagent.

A further embodiment of the present invention is a method and/or assayfor screening and/or identifying a test agent for the prevention and/ortreatment of TiLV comprising contacting or incubating a test agent to anucleotide comprising the nucleic acid sequence of SEQ ID NOs: 1-11 orfragments or variants thereof as described herein, and determining ifthe test agent binds to the nucleotide, wherein if the test agent bindsto the nucleotide, the test agent is identified as a therapeutic and/orpreventative agent for TiLV.

A further embodiment of the present invention is a method and/or assayfor screening and/or identifying a test agent for the prevention and/ortreatment of TiLV comprising contacting or incubating a test agent to anucleotide comprising the nucleic acid sequence of SEQ ID NOs: 1-11 orfragments or variants thereof as described herein, and detecting theexpression of the nucleotide before and after contact or incubation withthe test agent, wherein if the expression of the nucleotide is decreasedafter the contact or incubation with the test agent, the test agent isidentified as a therapeutic and/or preventative agent for TiLV.

A further embodiment of the present invention is a method and/or assayfor screening and/or identifying a test agent for the prevention and/ortreatment of TiLV comprising contacting or incubating a test agent to agene construct comprising a nucleotide comprising the nucleic acidsequence of SEQ ID NOs: 1-11 or fragments or variants thereof asdescribed herein, and detecting the expression of the nucleotide in thegene construct before and after contacting or incubating the test agentwith the gene construct, wherein if the expression of the gene isreduced or decreased after contact with the test agent or compound, thetest agent is identified as a therapeutic and/or preventative agent forTiLV.

A further embodiment of the present invention is a method and/or assayfor screening and/or identifying a test agent for the prevention and/ortreatment of TiLV comprising transforming a host cell with a geneconstruct comprising a nucleotide comprising the nucleic acid sequenceof SEQ ID NOs: 1-11 or fragments or variants thereof as describedherein, detecting the expression of the nucleotide in the host cell,contacting the test agent with the host cell, and detecting theexpression of the nucleotide in the host cell after contact with thetest agent or compound, wherein if the expression of the nucleotide isreduced or decreased after contact with the test agent or compound, thetest agent is identified as a therapeutic and/or preventative agent forTiLV.

The expression of a nucleotide or gene can be determined using ameasurable phenotype, either one that is native to the gene or one thatis artificially linked, such as a reporter gene.

A further embodiment is a method and/or assay for screening and/oridentifying a test agent for the prevention and/or treatment of TiLV,comprising contacting or incubating the test agent with a polypeptideencoded by a nucleotide sequence of SEQ ID NOs: 1-11, or a fragment orvariant thereof, and detecting the presence of a complex between thetest agent and the polypeptide, wherein if a complex between the testagent and the polypeptide is detected, the test agent is identified as aprevention and/or treatment for TiLV.

A further embodiment is a method and/or assay for screening and/oridentifying a test agent for the prevention and/or treatment of TiLV,comprising contacting or incubating the test agent with a polypeptideencoded by a nucleotide sequence of SEQ ID NOs: 1-11, or a fragment orvariant thereof and a known ligand of the polypeptide, and detecting thepresence of a complex between the test agent and the ligand, wherein ifa complex between the test agent and the ligand is detected, the testagent is identified as a prevention and/or treatment for TiLV.

Another embodiment of the present invention is a method and/or assay forscreening and/or identifying a test agent for the prevention and/ortreatment of TiLV, comprising contacting or incubating the test agentwith a polypeptide encoded by a nucleotide sequence of SEQ ID NOs: 1-11,or a fragment or variant thereof and a known antibody of thepolypeptide, and detecting the presence and quantity of unboundantibody, wherein the presence of the unbound antibody indicates thatthe test agent is binding to the polypeptide, and the test agent isidentified as a prevention and/or treatment for TiLV.

High throughput screening can also be used to screen the test agents.Small peptides or molecules can be synthesized and bound to a surfaceand contacted with the polypeptides, and washed. The bound peptide isvisualized and detected by methods known in the art.

The invention also provides for polypeptides to be used for rationaldrug design where structural analogs of biologically active polypeptidescan be designed. Such analogs would interfere with the polypeptide invivo, such as by non-productive binding to target. In this approach thethree-dimensional structure of the protein is determined by any methodknown in the art including but not limited to x-ray crystallography, andcomputer modeling. Information can also be obtained using the structureof homologous proteins or target-specific antibodies.

Using these techniques, agents can be designed which act as inhibitorsor antagonists of the polypeptides, or act as decoys, binding to targetmolecules non-productively and blocking binding of the activepolypeptide.

The term “agent” as used herein means a substance that produces or iscapable of producing an effect and would include, but is not limited to,chemicals, pharmaceuticals, biologics, small organic molecules,antibodies, nucleic acids, peptides, and proteins.

EXAMPLES

The present invention may be understood by reference to the followingnon-limiting examples, which are presented in order to more fullyillustrate the preferred embodiments of the invention. They should in noway by construed to limit the broad scope of the invention.

Example 1—Materials and Methods Used for Examples 2-9

Cell Culture: Eight established fish cell lines were used in this study:(1) CHSE-214 (ATCC CRL 1681) from the Chinook salmon Oncorhynchustshawytscha; (2) BF-2 (ATCC CCL 91), derived from the bluegill Lepomismicropterus; (3) BB (ATCC CCL 59), from the brown bullhead Ictalurusnebulosus; (4) and (5) EPC (ATCC CRL 2872 and KF-1), from the commoncarp C. carpio; (6) RTG-2 (ATCC CCL 55), from the rainbow trout Salmogairdneri; (7) FHM (ATCC CCL 42), from the fat head minnow Pimephalespromelas; and (8) E-11, from the striped snakehead Ophicephalus striatus(generously provided by M. Ucko, Israel Oceanographic and LimnologicalResearch). In addition, a culture of primary tilapia brain cells wasgenerated, developed as previously described in Hasegawa et al. 1997 andHedrick et al. 2000. Briefly, commercial Nile tilapia (O. niloticus) (50grams) were euthanized by anesthetic overdose (600 mg/liter tricainemethanesulfonate [MS-222]; Finquel, USA), and the brains were removedaseptically. The minced brains were manually homogenized and passedthrough a 100μ mesh grinders. The cells were then washed and seeded in12.5-ml sealed flasks (Becton-Dickinson, San Francisco, Calif., USA) at25° C. The initial culture medium contained 80% Leihovitz (L-15) medium(Gibco, USA), 10% inactivated fetal calf serum (FCS) (Gibco), and 10%inactivated tilapia serum medium, supplemented with L-glutamine (300mg/liter), HEPES (1%), penicillin (100 □g/ml), streptomycin (100 □g/ml),and amphotericin B (0.25 □g/ml).

During the first 21 days of incubation, 50% of the media were changedevery week. Thereafter, the monolayers were trypsinized and transferredinto new 25-ml flasks (Cellstar; Greiner Bio-One, Germany) with a 1:1mixture of conditioned medium (from old cultures) and fresh medium. Thecultures of the primary cells were passaged every other week. After 35passages, the tilapia serum and the condition media were omitted, andthe cells were split (at a ratio of 1:2) every 2 to 3 weeks in regularmedium (L-15 with 5% inactivated FCS).

Viruses and Virus Culture:

A total of 25 tilapia lake virus (TiLV) isolates were collected fromsuspected outbreaks that occurred between May 2011 and June 2013. Theisolations were obtained from all Israeli regions where fish arecommercially cultured: the coastal shore (2 isolations); the JordanValley (comprising the Bet-Shean Valley and the Yizrael Valley; 9isolations); and Upper and Lower Galilee (3 isolations). In addition, 11isolations were obtained from various species of wild tilapines from theSea of Galilee. An outbreak of farmed fish was defined as a sudden andunexplained rise in mortality (2% or more daily) for at least threeconsecutive days. If two wards were simultaneously affected on the samefarm, these were classified as a single outbreak. Therefore, eachisolate represents a distinct clinical outbreak. Viruses from wild fishdisplaying ocular lesions were isolated from commercially caught fish inthe Sea of Galilee; each isolate represents a different catch. Fishweighing 20 to 200 grams or 40 to 350 grams (wild and farmed fish,respectively) were collected during the hot seasons (May to October;water temperature, between 22 and 32° C.). To minimize contaminationrisks, the brains and viscera (kidneys, livers, spleens, and hearts) ofthe suspected fish were removed aseptically, pooled, and manuallyhomogenized with nine volumes of Hanks' balanced salt solution (HBSS),centrifuged at 3,000×g for 10 minutes, and the supernatants werefiltered through 0.22 μl filters (Sarstedt, Germany). The filtrates werestored at −80° C. until use. For infection, monolayers (about 90%confluence) were washed twice with HBSS and incubated with 500 μl of thevirus filtrate at 25° C. for 1 hour, after which the cells were washedwith HBSS, supplemented with L-15 medium (2% FCS), and incubated at 25°C. The cultures were observed daily for 21 days for cytopathic effects(CPE). In experiments where the tilapine disease was reproduced by virusinjection, a virus named TiLV×2 was also used, which was purified by twosuccessive rounds of endpoint dilution assays. This was performed withE-11 cultures, infected with serial dilutions of TiLV (isolate 4/2011;obtained from the brain of a diseased St. Peter's fish that wascollected from the Kinneret Lake on June 2011).

Titration of Virus:

The original virus-containing culture supernatant (isolate 4/2011) wascultured in E-11 cells and serially diluted in 10-fold increments withHBSS and 50 μl from each dilution was inoculated onto E-11 monolayers in96-well plates. Four wells were used for each diluted sample. The plateswere incubated at 25° C. and observed daily for CPE. After 7 days, the50% tissue culture infectious dose (TCID₅₀) (ml⁻¹) was calculated by themethod of Reed and Muench 1938.

Electron Microscopy Analyses:

For examinations of TiLV by transmission electron microscopy,E-11-infected cultures were scraped from the flask, centrifuged (2,000rpm for 7 minutes), fixed with 1.5% glutaraldehyde in 0.1 M sodiumcacodylate (pH 7.2) for 2 hours, and then rinsed five times in phosphatebuffer (pH 7.2). The pellets, consisting of infected E-11 cells, werepost-fixed in 1% OsO₄ in phosphate buffer and dehydrated with increasingconcentrations of ethanol. The pellets were then washed twice with 100%propylene oxide and treated with propylene oxide-Epon (3:1) for 30minutes, followed by propylene oxide-Epon (1:1) for 15 minutes. Finally,the pellets were embedded in 100% Epon and left overnight. Thin sections(70 to 90 nm) were placed on Formvar-coated copper grids and stainedwith uranyl acetate, followed by lead citrate, according to the Reynoldsmethod (Reynolds 1963). All micrographs were taken with a JEOL 1200-EXelectron microscope operating at 60 or 80 kV (Electron Microscopy [EM]Unit, institute of Biotechnology, Bar-Ilan University, Israel). EManalysis of the negatively stained virion pellets was carried at theEMUnit, Tel Aviv University, exactly as described before in Oberpichleret al. 2008, with an A JEM 1200-EX transmitting electron microscope(JEOL-USA, Peabody, Mass., USA). The virions for this analysis werepelleted by ultracentrifugation through 25% sucrose cushions.

Purification of Virus from Culture Supernatants Using Sucrose GradientFractionation:

Cultured E-11 cells were infected with TiLV (isolate 4/2011), and theculture supernatant was cleared from the cell debris by centrifugation(10 minutes at 3,000 rpm). The supernatant was layered onto 2 ml of a30% (wt/vol) sucrose-Tris-EDTA (TE) buffer cushion and centrifuged for 2h in a T865 rotor at 65,000 rpm (Sorvall Discovery 90SE). The pellet wasresuspended in TE buffer and layered onto a sucrose step gradient(Bacharach et al. 2000; Laham and Bacharach 2007; Melamed et al. 2004).The gradient consisted of 3-ml layers with sucrose concentrations of 70,60, 50, 40, 30, 20, and 10% (wt/vol) in TE, from bottom to top.Ultracentrifugation was performed in a TST41.14 rotor for 2 hours at40,000 rpm (Sorvall Discovery 90SE). One-milliliter fractions were takenfrom the top of the gradient, and the virions were pelleted from eachfraction by ultracentrifugation (for 2 hours at 65,000 rpm; T865 rotor;Sorvall Discovery 90SE) and resuspended in 1 ml of TE buffer. 100 μlaliquots from each sample were incubated with naive E-11 cells tomonitor for CPE. The incubation of cultures with negative controls,consisting of aliquots from fractions of an identical sucrose gradienthut with no addition of culture supernatants, resulted in no CPE.

Isolation of Nucleic Acids from Purified Virions and cDNA Synthesis:

Nucleic acids were extracted from purified virion pellets using peqGOLDTrifast for RNA (Peqlab, Germany) or the High Pure PCR templatepreparation kit for DNA (Roche, Germany). Reverse transcription wasperformed with the Verso cDNA kit (Thermo, Lithuania), according to themanufacturer's instructions. To identify TiLV-specific sequences, thesupernatants of TiLV (isolate 4/2011)-infected E-11 cultures werecleared from the cell debris by centrifugation (for 10 minutes at3,000×g), and the purified supernatants were subjected to furtherpurification by ultracentrifugation (for 2 hours in a T865 rotor at65,000 rpm [Sorvall Discovery 90SE]) through a 30% sucrose cushion. Thepellet was resuspended in TE, and virions were further purified bysucrose cushions of 40 to 70% (wt/vol). After ultracentrifugation(TST41.14 rotor for 2 hours at 40,000 rpm; Sorvall Discovery 90SE), the40% sucrose fraction was collected, and virions were pelleted byadditional ultracentrifugation (TST41.1 rotor for 2 hours at 40,000 rpm;Sorvall Discovery 90SE). RNA was extracted from the pellets by guanidinethiocyanate (peqGOLD Trifast; Peqlah). cDNA was generated by reversetranscription and random priming, using the purified RNA as a template.The fragments of this cDNA were isolated by shotgun cloning (Nehls andBoehm 1995).

Shotgun Cloning by Random Priming:

Shotgun cloning was performed as described by Nehls and Boehm. Thepurified cDNA (approximately 10 ng) was double primed with MluI(N)6primer (GGAACTCAATGCACGCGTNNNNNN) (SEQ ID NO: 13) using RcddyMix PCRmaster mix (Thermo, Lithuania). The primed products were amplified byPCR with MluI primer (GGAACTCAATGCACGCGT) (SEQ ID NO: 14) and clonedinto the pJET1.2/blunt vector (CloneJET; Fermentas/Thermo, Lithuania),which was transformed into Escherichia coli strain HIT-DH5a cells (RealBiotech, Taiwan). Ampicillin-resistant transformants, grown at 37° C. onLB agar plates containing 100 μg/ml ampicillin, were picked and grownovernight in 5 ml of LB supplemented with 100 μg/ml ampicillin. PlasmidDNA was isolated using the HiYield plasmid minikit (RBC, Taiwan). Theinserts were amplified by PCR using the pJET1.2-derived primers,separated by electrophoresis in a 1.0% gel, placed in 1×Tris-acetate-EDTA (TAE) buffer at 80 V for 1.5 hours, stained withethidium bromide, excised, and gel purified using the GeneJET gelextraction and DNA cleanup micro kit (Thermo, Lithuania). Singlefragments were sequenced by Hy Laboratories (Israel) using ABI 3730. Thesequences were analyzed for homologies to nucleotide sequences in theGenBank database using the nucleotide Basic Local Alignment Search Tool(BLASTn) and the Vector NTI 6 (InforMax, Inc.) software. Furthersearches of protein databases were done by BLASTx. The internal primersfrom each sequenced clone were tested for PCR amplification of the TiLVgenome. The primers derived from clone 7450 specifically amplified thecognate sequence from TiLV-infected cultures, in reversetranscription-PCRs (RT-PCRs).

Rapid Amplification of cDNA Ends (RACE):

To extend the sequence of clone 7450 obtained by shotgun cloning, 3′ and5′ rapid amplification of cDNA ends (RACE) reactions were carried out asdescribed before (Scotto-Lavino et al. 2006a; Scott-Lavino et al.2006b), using total RNA that was extracted from TiLV-infected E11 cellsby EZ-RNA reagent (Biological Industries). Briefly, for 3′ RACE, cDNAwas generated using primer Q_(T).(CCAGTGAGCAGAGTGACGAGGACTCGAGCTCAAGCTTTTTTTTTTTTTTTTTVN) (SEQ ID NO: 15)and the SuperScript III first-strand synthesis system for RT-PCR(catalog no. 18080-051; Invitrogen), according to the manufacturer'sinstructions. The cDNA was amplified with clone 7450/150R primer(TATCACGTGCGTACTCGTTCAGT) (SEQ ID NO: 16) that was derived from aninternal sequence of the shotgun fragment, and with Q_(O) primer(CCAGTGAGCAGAGTGACG, (SEQ ID NO: 17) derived from Q_(T) primer), usingEx-Taq enzyme (catalog no. RR001A; TaKaRa). The resulting PCR productswere diluted 1:20 and were subjected to a second PCR with the nestedprimers Q_(I) (GAGGACTCGAGCTCAAGC (SEQ ID NO: 18), derived from Q_(T)primer) and E11-inf-R (AAGTTCTCTTGCCTCTTGG (SEQ ID NO: 19), derived fromthe sequence of the shotgun fragment). For 5′ RACE, cDNA was generatedas above but with primer clone 7450/150F (CACCCAGACTTGCGGACATA) (SEQ IDNO: 20). Poly(A) tails were added to the cDNA using terminal transferase(catalog no. 3333566; Roche), according to the manufacturer'sinstructions. The tailed cDNA was amplified by PCR using primerE-11-inf-F (TCCAAGGAAACAGCTGAGC (SEQ ID NO: 21), derived from thesequence of the shotgun fragment), together with a mixture of the Q_(O)and Q_(T) primers. The resulting PCR products were diluted 1:20 andsubjected to a second nested-PCR using the E11-inf-F-in(GAGGCAATATGGATTCTTCG) (SEQ ID NO: 22) and Q₁ primers.

RT-PCR:

Samples from the brain, heart, head kidney, spleen, and liver were takenfrom clinical cases of suspected TiLV outbreaks, pooled, and directlyfrozen at −80° C. Total RNA was purified using peqGOLD Trifast (Peqlab,Germany), according to the manufacturer's instructions, followed byreverse transcription and amplification (Verso 1-step RT-PCR ReddyMixkit; Thermo, Lithuania). The random primers of the kit were substitutedwith the external specific primer ME1 (GTTGGGCACAAGGCATCCTA) (SEQ ID NO:23) and clone 7450/150R (TATCACGTGCGTACTCGTTCAGT) (SEQ ID NO: 16).Cycling was performed at 50° C. for 15 minutes (reverse transcription),95° C. for 2 minutes (enzyme inactivation), and 35 cycles at 95° C. for30 seconds, 56° C. for 60 seconds, and 72° C. for 60 seconds; thereaction was terminated by 72° C. for 7 minutes. The PCR products wereresolved in 1% agarose gels in 0.5×TAE buffer (40 mM Tris-acetate and 1mM EDTA).

Nuclease Sensitivity Assays:

The supernatant (9 ml) of a TiLV-infected E-11 culture was collected,and virions were purified and pelleted through a 25% (wt/vol) sucrosecushion, using ultracentrifugation (107,000×g at 4° C. for 2 hours). Asupernatant of uninfected E-11 culture was used as a control. To digestfree nucleic acids, the pellets were resuspended in 300 μl of 1× DNasebuffer (10 mM Tris-HCl [pH 7.5], 2.5 mM MgCl₂, and 0.5 mM CaCl₂) andwere supplemented with 33 μg of RNaseA (Sigma R4642) and 1 U DNase(Baseline-ZERO DNase). The samples were incubated for 40 minutes at roomtemperature, after which each reaction mixture was diluted in 9 nil ofLeibovitz (L-15) medium, supplemented with 5% FCS, and virions werepelleted as described above. To release nuclease protected nucleic acidsfrom the virions and to digest possible leftovers of RNase A and DNaseI, the pellets were resuspended in 150 μl of proteinase K buffer (50 mMTris-HCl [pH 7.5], 100 mM NaCl, 10 mM EDTA, 1% SDS), supplemented with100 μg/ml proteinase K (Roche), and the proteins were digested for 30minutes at 37° C. The nucleic acids were extracted byphenol-chloroform-isoamyl alcohol (CIP) and were precipitated withethanol, 0.3 M sodium acetate (pH 5.2), and glycogen as a carrier. Thenucleic acids were resuspended in 20 μl of buffer (10 mM Tris-HCl [pH8.3], 10 mM MgCl₂, 1 mM dithiothreitol [DTT], 60 mM NaCl), and 3 μl wasadded to 100 μl of RNase I buffer (100 mM NaCl, 50 mM Tris-HCl [pH 7.9],10 mM MgCl₂, 1 mM DTT) with or without 50 units of RNase 1 (catalog no.M02435; NEB). Digestion was carried out for 5 minutes at 37° C., and thenucleic acids were CIP extracted and precipitated as described above.The nucleic acids were resuspended in 20 μl of reverse transcriptionreaction mixture; a reaction without reverse transcriptase was alsoassembled to ensure the absence of protected DNA. The resulting cDNA wasamplified with TiLV-specific primers (Nested ext-1[TATGCAGTACTTTCCCTGCC] (SEQ ID NO: 24) and Nested ext-2[TTGCTCTGAGCAAGAGTACC] (SEQ ID NO: 25)) or with snakehead retrovirus(SnRV)-specific primers (Snakehead gag-pol fw [CAGATCACTGATCGATGC] (SEQID NO: 26) and Snakehead gag-pol rev [GTCTGAAAGGTAAGGTGG] (SEQ ID NO:27)). The amplified products (491 and 284 bp for TiLV and SnRV,respectively) were separated by electrophoresis in 1% agarose gels.

Ether and Chloroform Sensitivity Assays:

TiLV sensitivity assays for ether and chloroform were performed asdescribed before (Crandell et al. 1975; Hutoran et al. 2005).

Experimental Reproduction of the Disease and Ethical Issues:

The tilapine species used in this study, O. niloticus (strainChitralada), was grown at a specific-pathogen-free (SPF) facility(UV-treated pathogen free environment) at a constant temperature of 28°C. The fish were fed a daily regimen of 2% (wt/wt); the water parameters(O₂>5 ppm, NH4+<1 ppm, NaCl<1 ppt) were kept constant. Allexperimentally induced infections were carried out with the fieldisolate of TiLV (isolate 4/2011, passage 2), which was aliquoted andkept frozen at −80° C. Before use, the virus was thawed and culturedonce more (passage 3). For artificial reproduction of the disease,2.6×10⁵ TCID₅₀ was injected intraperitoneally (i.p.) (group 1) into eachfish (30 to 35 grains). All experiments were carried out in triplicatewith groups of 30 fish. To prevent waterborne infection, each group offish was kept in a separate 100-liter aquarium. During the cohabitationtrials (group 2), groups of 30 fish were kept in 200-liter aquariumsthat were divided into three compartments by water-permeable grids,which allowed water (but not fish) circulation throughout the aquarium;a control group was kept in the middle. The fish surviving primary i.p.infection were pooled and 3 weeks after were divided into two groups(each with 15 fish) and infected once again by i.p. injection. Thecontrol groups were injected with uninfected (naive) E-11 cultures.

When in vivo/ex vivo experiments were conducted, the brains ofindividual TiLV-injected fish were collected (5 to 7 dayspost-injection) and minced as above. The homogenates (500 μl) wereincubated with confluent E-11 cultures. Upon CPE appearance, thesupernatants were collected and injected (200 μl) i.p. into naive fish.

The health conditions of the fish were carefully monitored throughoutthe growing and experiment periods; external signs and mortality rateswere monitored twice daily for a total of 21 days. The animal care,experimental handling, and safety regulations conformed to theguidelines established by the Committee on Laboratory Animal Care at theIsraeli Veterinary Services and were conducted under permit 020_b5471_6,issued by the Israeli Committee for Animal Welfare.

Histological Analysis:

Tissue samples were collected from euthanized naturally infected fish byabdominal incision and were fixed in 10% neutral buffered formalin. Thespecimens were embedded in paraffin (Paraplast Plus; Diapath), cut bymicrotome (Reichert-Jung 2050) into serial 5 μm sections, stained withhematoxylin and eosin (H&E) 277 (Roberts et al. 2012), and examinedunder a light microscope (Leica DMRB). Images were acquired by a Nikondigital light system.

Statistical Analysis:

The results of the in vivo experiments were presented as percentages ofthe mean mortality rates from three (or two, in case of surviving fish)independent experiments. Each experiment included three experimentalgroups (three independent repeats) of 30 fish.

The experiments with the surviving fish were performed in duplicate (twoindependent repeats), in which each group was composed of 20 fish.Variability between the experiments (infection by direct intraperitonealinjection, infection by cohabitation, and control fish) was determinedby chi square tests, in which a P value of greater than 0.05 wasconsidered significant.

Culture Deposition:

TiLV (CNCM accession no. 1-4817) was deposited at the CollectionNationale de Cultures de Microorganismes (CNCM) at the Institut Pasteur,Paris, France.

Nucleotide Sequence Accession Number:

The GenBank accession number for the extended sequence of clone 7450 isKJ605629 (SEQ ID NO: 1).

Example 2—Geographical Distribution and Characteristics of DiseasedTilapines from the Sea of Galilee and Commercial Ponds

Disease outbreaks in wild and commercial tilapines were detected in theSea of Galilee and in commercial ponds in Israel, located in theNorthern coastal shore, Bet-Shean, Yizracl, the Jordan Valley, and Upperand Lower Galilee. In commercial ponds, this disease resulted in massivemortality (FIG. 1A). The sampling of fish from commercial catches at theSea of Galilee revealed that all tilapine species are susceptible to thedisease, although mass mortalities were not observed. In this case, thediseased fish presented with pronounced ocular lesions (FIG. 1B).

Pathological findings included gross lesions characterized mainly byocular alterations, including opacity of the lens (cataract). Inadvanced cases, the lesions included ruptured lenses with phacoclasticinduced uveitis or endophthalmitis accompanied by the formation of acyclitic membrane, followed by swelling of the eyeball (buphthalmia),loss of globe integrity with occasional perforated cornea and poring ofinspissated content or shrinkage, and loss of ocular functioning(phthisis bulbi) (FIG. 1B). Other lesions included skin erosions(observed in diseased pond-raised tilapines; FIG. 1C), hemorrhages inthe leptomeninges, and moderate congestion of the spleen and kidney(FIG. 1D).

The histologic lesions of the brain included edema, focal hemorrhages inthe leptomeninges, and capillary congestion in both the white and graymatter (FIG. 1E). Foci of gliosis and occasional perivascular cuffs oflymphocytes were detected (FIG. 1F). Some neurons within thetelencephalon and particularly in the optic lobes displayed variouslevels of neuronal degeneration, including cytoplasmic rarefaction andvacuolation and peripherally displaced nuclei (central chromatolysis).

Ocular lesions included an undulated, thin, and frequently coiled andruptured lenticular capsule, surrounded by circular fibroplasia andfibrosis with multiple synechiae to the iris and ciliary body (posteriorsynechia) and moderate numbers of eosinophilic granulocytic cells andmclano-macrophage centers or MMCs. The infiltrate extended into theanterior chamber, iris, vitreous humor, and choroid (endophthalmitis).There were cataractous changes within the lens characterized byeosinophilic homogenous spherical structures (morgagnian globules),markedly enlarged lens epithelial cells with abundant eosinophilicmicrovacuolated cytoplasm (bladder cells), large lakes of proteinaceousfluid (liquefied lens fibers), mineralization, and flattened elongatedcells (fibrous metaplasia) (FIG. 1Ga, compared to normal lens in FIG.1Gb). The squamous epithelium of the cornea was frequently eroded andulcerated and infiltrated by moderate numbers of lymphocytes,macrophages, and eosinophilic granulocytic cells, and it was underlinedby stromal neovascularization and edema (FIG. 1H).

The hepatic parenchyma displayed occasional randomly distributed foci ofhepatocellular swelling and clearing, with cytoplasmic accumulation ofgranular yellow to brown pigment. The spleen was hyperplastic, withproliferating lymphocytes surrounding the ellipsoids. MMCs wereincreased in size and number in both the liver and the spleen. MMCs aredistinctive clusters of pigment-laden cells, commonly seen within thereticuloendothelial supporting matrix of hematopoietic tissues. MMCproliferation is associated with late stages of chronic infection as aresponse to severe tissue injury in a variety of infections (especiallyviruses) or poor environmental conditions. Therefore, they areconsidered indicators of fish population health (Agius and Roberts2003).

Example 3—Isolation of the Etiological Agent from Infected Specimens

To culture potential pathogens from diseased tilapines, the organs offish with the characteristics described above were pooled, homogenized,and incubated with eight different cell lines as described in Example 1.No known pathogen was identified, and only the established E-11 cellline and the primary tilapia brain cells consistently showed CPE uponincubation with the above-mentioned homogenates. In E-11 cells, CPEbecame visible 5 to 7 days postinoculation, with the appearance ofcytoplasmic vacuoles and plaque formation (FIG. 2A), which rapidlyprogressed to an almost-complete disintegration of the cell monolayer(at 9 to 10 days postinoculation). The CPE in primary tilapia braincells was characterized by conversion of the typical elongated cellsinto swollen, rounded, and granulated cells, which were clearly observedat 10 to 12 days postinoculation (FIG. 2B), leading to vast monolayerdetachment (days 14 to 19) but without plaque formation. The controlmock-infected E-11 and primary tilapia brain cultures did not show anyCPE (FIGS. 2C and 2D, respectively). Similar results were obtained whenthe supernatants of the cultures with CPE were used to inoculate naivecultures (tested for up to 18 passages) and when the supernatants, orthe above-mentioned homogenates, were filtered through 0.22 □m filters.In addition, the number of plaques induced by the agent was directlyrelated to its dilution, yielding a one-hit curve. A single infectiousunit was therefore sufficient to produce a plaque. These resultsindicated that the described CPE was due to the presence of aninfectious agent, likely a virus. The CPE-causing agent was recoveredfrom 25 samples, collected from all Israeli regions where fish werecultured.

Example 4—Morphological Features of Virus-Like Particles

Further support for viral infection in E-11 cultures showing CPE camefrom EM examination of thin sections of these cells as described inExample 1. This analysis revealed the presence of sparse electron-denseparticles (diameter, 55 to 75 nm), enclosed in the intracytoplasmicmembrane (FIG. 2E) or within the cytoplasm (FIG. 2F). No such particleswere found in the healthy control cell cultures. Of note, theseparticles do not originate from the snakehead retrovirus (SnRV) that isexpressed in E-11, since assemblies of this C-type retrovirus are largerand are generated only at the plasma membrane; moreover, SnRV nascentvirions were not visualized by EM in this specific cell line (althoughSnRV sequences could be amplified by PCR).

Pellets, purified from the supernatants of infected E-11 cultures byultracentrifugation through 25% sucrose cushions, were negativelystained and examined by EM. This analysis revealed virion-likestructures (approximately 75 to 80 nm) surrounded by a readily detectedthick coat (FIG. 2G). Such virions were abundant and were not detectedin the control pellets prepared from naive E-11 cells.

Example 5—Sensitivity of the Infectious Agent to Ether or Chloroform

The EM analyses from Example 4 indicated that the infectious agent,isolated from diseased tilapia, is an enveloped virus. To test this,virions in the supernatants of E-11-infected cells were exposed toeither ether or chloroform and the effect of these treatments oninfectivity was measured as described in Example 1.

Table 1 summarizes the results of two and three repeats of the ether andchloroform sensitivity assays, respectively. A reduction in theinfectivity of approximately three (chloroform) to five (ether) ordersof magnitude was observed, demonstrating the sensitivity of the agent tothese solvents. This indicated that the infectious agent is indeedenveloped by a lipid membrane.

TABLE 1 Ether and chloroform sensitivity assays TCID₅₀/ml TCID₅₀/mlAverage Expt without plus Fold fold Treatment no. treatment treatmentreduction reduction Ether 1 10^(4.49) 10⁻¹ 10^(5.49) 1.58 × 10⁵ 210^(4.83) 10 10^(3.83) Chloroform 1 10^(4.63) 10^(1.5) 10^(3.15) 0.93 ×10³ 2 10^(3.5) 10^(1.5) 10² 3 10^(4.63) 10^(1.5) 10^(3.23)

Example 6—Initial Molecular Characterization of Tilapine Virus

Having demonstrated that an infectious agent can be isolated fromdiseased fish and can be propagated in specific cell culture (Examples3), this disease-causing agent was named tilapia lake virus (TiLV), as areference to the site from which it was initially isolated.

To purify TiLV, TiLV-infected culture supernatants were fractionatedthrough velocity sucrose step gradients ranging from 10 to 70% sucrose.The CPE-inducing activity was mainly localized to the 30 to 40% sucrosefractions.

To further identify TiLV-specific sequences, RNA was extracted from TiLVvirions (purified by ultracentrifugation through sucrose cushions) andused as a template in a reverse transcription reaction. The fragments ofthe resulting cDNA were cloned using a shotgun approach as described inExample 1. This approach allows the cloning of cDNAs that are present atsmall amounts, without prior knowledge of their sequences. One of thesefragments (clone 7450) was subjected to 5′ and 3′ RACE reactions,resulting in the identification of 1,326 bases of a TiLV sequence (SEQID NO: 1) (GenBank accession no. KJ605629), which contained an openreading frame (ORF) of 420 amino acids (SEQ ID NO: 12). SEQ ID NO: 1 waslater identified to be part of SEQ ID NO: 9.

No significant homologies were found in both the nucleic acids andprotein sequences of this clone using BLAST searches (Altschul et al1997; Johnson et al. 2008) in the GenBank databases.

Example 7—PCR for TiLV Detection

As described in Example 1, PCR was performed to detect TiLV. Toestablish a PCR assay for detecting TiLV, total RNA was extracted fromthe brains, kidneys, hearts, livers, and spleens of moribund fish. Inaddition, RNA was extracted from TiLV-infected primary tilapia brain orE-11 cultures and was used as a template for cDNA generation. Thesesamples were subject to RT-PCR with primers that were derived from clone7450 (SEQ ID NO: 1). A 250-bp fragment was amplified with ME1(GTTGGGCACAAGGCATCCTA) (SEQ ID NO: 23) and clone 7450/150R(TATCACGTGCGTACTCGTTCAGT) (SEQ ID NO: 16) primers. The PCR assaysresulted in the amplification of the expected 250-bp fragment from thebrains of TiLV-infected fish (FIG. 3A).

The amplification of TiLV was achieved only after a reversetranscription step, even under conditions in which the samples were nottreated with DNase (FIG. 3B). Total RNA was extracted from thesupernatant or from cell extracts of TiLV-infected E-11 culture, or fromnaive E-11 culture. The samples were not treated with DNase, and reversetranscription was carried out (+) or not (−) prior to the PCR step. A“no RNA” negative control (lane 7) was also included. A 491-bp fragmentwas amplified with the primers Nested ext-1 (TATGCAGTACTTTCCCTGCC) (SEQID NO: 25) and Nested ext-2 (TTGCTCTGAGCAAGAGTACC) (SEQ ID NO: 26), onlywhen reverse transcriptase was carried out. This is highly indicative ofan RNA genome for TiLV. Consistent amplification in the samples of braintissues was observed compared to the other organs. Amplification wasalso observed in TiLV-infected primary tilapia brain and E-11 culturesbut not in a negative control that included cDNA prepared from the brainof a healthy (naive) fish (FIG. 3B).

No amplification was observed in additional negative controls, whichincluded mock-infected primary tilapia brain and E-11 cultures, or E-11cultures infected with the viral nervous necrosis (VNN) betanodavirus.Of note, the absence of amplification in the sample of VNN-infectedcells further indicates that clone 7450 represents a sequence derivedfrom TiLV rather than a fish gene that is upregulated upon infection. Inall cases, sequencing of the amplified fragments revealed full identitywith the expected sequence.

The above PCR assay was also exploited to further test the RNA nature ofthe TiLV genome. For this, the virions in the supernatants ofTiLV-infected E-11 cultures were exposed to DNase I and RNaseA to digestnucleic acids that are not protected by virions. The particles were thenpelleted through sucrose cushions and digested with proteinase K, andthe protected deproteinized nucleic acids were purified. These nucleicacids were exposed to RNase I, an enzyme with a preference forsingle-stranded RNA. The resulting products were reverse transcribed ornot, and subjected to PCR amplification using TiLV-specific primers.(Nested ext-1 [TATGCAGTACTTTCCCTGCC] (SEQ ID NO: 25) and Nested ext-2[TTGCTCTGAGCAAGAGTACC] (SEQ ID NO: 26); or SnRV-specific primers(Snakehead gag-pol fw [CAGATCACTGATCGATGC] (SEQ ID NO: 27) and Snakeheadgag-pol rev [GTCTGAAAGGTAAGGTGG] (SEQ ID NO: 28).

The amplification of TiLV sequences was observed only after a reversetranscription step (FIG. 3C, lanes 1 and 3, similar to the result inFIG. 3B), and only if RNase I was avoided (FIG. 3C, lanes 1 and 2). Thesingle-stranded genomic RNA of SnRV, which was copurified along the TiLVgenome, was used as an internal positive control for this assay (FIG.3C, lanes 5 to 8). These results further indicated that the TiLV genome,encapsidated in the virion, is composed of RNA in single stranded form.

Example 8—Reproduction of Tilapine Disease by Intraperitoneal Injection

To test if TiLV can cause disease in tilapines, the supernatants fromnaive or TiLV-infected E-11 or primary tilapia brain cultures werefiltered (0.22 □m), and 200 □l was injected i.p. into naive Tilapianilotica (groups of 30 fish as described in Example 1). All of the naivefish that were inoculated with the control supernatants (of naive E-11cultures) remained asymptomatic. However, 74 to 85% of the fish thatwere injected with the supernatants of TiLV-infected E-11 orTiLV-infected primary tilapia brain cultures developed clinical disease(lethargy, discoloration, ocular alterations, skin patches, andulcerations) and died within 10 days (FIG. 4). The same mortality ratewas also observed for fish injected with TiLV that was purified byendpoint dilution assay (TiLV×2). Furthermore, the brains fromexperimentally infected fish were harvested and co-incubated with naiveE-11 cells. Such cultures developed a characteristic CPE. Thesupernatants of these cultures were then harvested and injected intonaive fish, resulting in the appearance of the disease in fish. Overall,this in vivo/ex vivo passage experiment was serially repeated threetimes, with a consistent mortality rate of 75 to 85% in each roundwithin 10 days post-injection.

This clearly confirmed that TiLV, isolated from infected fish andpropagated in E-11 cells, was indeed the etiologic agent of the disease.Importantly, fish that survived the experimentally induced disease (35fish) were completely immune to disease development upon a challengeconsisting of a second i.p. injection (3 to 4 weeks after the firstinjection). This indicates that fish can mount a protective immuneresponse to TiLV.

Example 9—Reproduction of Tilapine Disease by Cohabitation

To determine if TiLV is transmissible in a setting resembling naturalconditions, a cohabitation experiment was performed in which naive fishwere cohabitated with fish experimentally infected with TiLV asdescribed in Example 1.

These experiments clearly demonstrated that the naive fish developed alethal disease, with a mortality rate similar to the one obtained by thei.p. route but with slower kinetics (2 to 3 days delay in reaching 50%mortality, P<0.05 (FIG. 4)). These experiments provide proof of theability of TiLV to spread through a waterborne route.

Example 10—Bioinformatic Data Analysis

Ion Torrent data were generated using as a template brain RNA of sicktilapia after depletion of ribosomal RNA (two libraries). Illumina datawere generated for nuclease treated and sucrose gradient purifiedparticles obtained from infected E11 culture cells (two libraries). IonTorrent libraries were preprocessed with cutadapt (Martin 2011) toremove low quality ends, trimmed to 150 bp maximum length, and strippedof adapter sequences.

Reads from all four libraries (2 brain and 2 cell culture) weretaxonomically classified using taxMaps(https://github.com/nygenome/taxmaps) by mapping against the NationalCenter for Biotechnology Information's (NCBI) nt database, the NCBIRefSeq database (Pruitt et al. 2012), the tilapia reference genomesequence (Orenil 1.1) and corresponding annotated tilapine mRNAsequences (Brawand et al. 2014). Unclassified reads (not mapping to anyknown sequence) were then independently assembled using the VICUNAassembler (Yang et al. 2012). Contigs from each library were alignedwith BLAST (Camacho et al. 2009) against all contigs from the other 3libraries, retaining hits with an e-value of 1e-10 or lower. Singlelinkage clustering was used to group together all the contigs thatshowed any similarity. On the assumption that the infectious agentshould be present in all 4 libraries, 10 contig clusters were identifiedthat contained at least one contig from each of the 4 libraries.

Within each cluster, contigs were aligned to each other and manuallyassembled to generate maximum length sequence after inverted tandemduplications at the ends of contigs, likely resulting from amplificationartifacts, were removed. Overlapping predicted ORFs in contigs fromdifferent assemblies were used to correct for frameshift errors (mostlyoccurring due to indels in the Ion Torrent reads) and to infer thelongest possible ORF.

Based on a model wherein the genomic segments are anticipated to containconserved termini, a combination of k-mer analysis, read depth analysis,and manual curation was used to build 5′ and 3′ terminal sequence motifsto refine terminal sequences. Mapping of the 10 final consensussequences against the initial raw read data with BWA-MEM (Li 2013)demonstrated that 99% of the unidentified reads from the Illuminalibraries and 87% of unidentified reads from the Ion Torrent librariesmapped to the consensus sequences.

Example 11—Complete Characterization of TiLV Genome

Based on the bioinformatics consensus sequences PCR primers weredesigned and contiguous sequences of all 10 clusters amplified frominfected Tilapia specimens. Terminal sequences of each of the genomesegments were revised by 5′- and 3′-Rapid Amplification of cDNA Ends(RACE) (Table 2). Analogous analyses by high throughput sequencing andPCR amplification was performed on samples from diseased Tilapia fromIsrael, and then performed on samples from Tilapia with a similardisease syndrome from Ecuador. Sequences for all 10 segments wereobtained that showed a greater than 94% nucleotide sequence identity tothe corresponding sequences from Israeli Tilapia.

Fragments of each segment were cloned into plasmid vectors to generateprobes for Northern blotting. Three mixes of probes were usedrepresenting segments 1, 4, 7, and 10 (Combo 1); 3, 6, and 9 (Combo 2);or 2, 5, and 8 (Combo 3) to prevent signal overlap from similar sizedsegments. Total RNA was extracted from the livers of diseased Ecuadoriantilapia and from the culture cells infected with brain derived TiLV fromIsraeli tilapia or cell culture supernatant. Northern blot demonstratedthe presence of all 10 sequences in nucleic acid extracts from infectedIsraeli and Ecuadorian Tilapia (FIG. 5).

Homology searches in the NCBI sequence database yielded only a singlehit for segment 1 that indicated very distant homology to orthomyxoviralRNA-dependent RNA polymerase motifs (Table 2).

TABLE 2 TiLV genome segments Homology to Size known Segment Cluster #[nt] sequences SEQ ID NO 1 5 1640 orthomyxoviral 7 PB1 polymerasedomains 2 0 1471 none 2 3 7 1371 none 9 4 4 1249 none 6 5 1 1098 none 36 2 1044 none 4 7 3 777 none 5 8 9 657 none 11 9 8 549 none 10 10 6 465none 8

Example 12—Diagnostic PCR

Conventional PCR: Primers NM-CLU7-SF1, 5′-AGT TGC TTC TCA YAA GCC TGC TA(SEQ ID NO: 28) and NM-CLU7-SR1, 5′-TCG TGT TCA CAR CCA GGT TTA CTT (SEQID NO: 29) were designed to amplify an approximately 245-nt region ofTiLV segment 3 (cluster 7, Accession no. KJ605629). cDNA fromTRI-reagent (Invitrogen) extracted RNA was synthesized with SuperscriptIII (Life Technologies) and random hexamers according to vendor'sprotocol. PCR was performed with Amplitaq Gold (Life Technologies) andPCR products visualized on agarose gels, purified with Purelink GelExtraction kit (Invitrogen), and confirmed for target specificity bySanger sequencing on both strands (GeneWiz, N.J.). (FIG. 6)

Quantitative Real-Time PCR:

A quantitative real-time PCR assay for TiLV was established targetingTiLV segment 1 and beta-actin as a house-keeping gene control(Oreochromis niloticus beta-actin mRNA, XM_003443127) (FIG. 7). For bothassays the respective target regions were cloned into plasmid vectorsusing primers TiLV-CLU5-cF1, 5′-GGT CAA TTC GAG TCA TGC TCG (SEQ ID NO:30)/TiLV-CLU5-cR1, 5′-GCT GGA CTG CTT TAT AAA TAG CAT AG (SEQ ID NO:31), or TIL-Actin-cF1, 5′-ATC CTG CGT CTG GAC CTG GCT (SEQ ID NO:32)/TIL-Actin-cR1, 5′-TGC CAA TGG TGA TGA CCT GTC (SEQ ID NO: 33) togenerate quantitative calibration standards (FIGS. 7A and 7B).

Targeted real-time PCR were performed using the following specificoligonucleotides:

TiLV segment 1 (cluster 5) CLU5-mRNA-qF1, (SEQ ID NO: 34)5′-AGC TAT GTT ATC TGG CGC T CLU5-mRNA-qR1, (SEQ ID NO: 35)5′-GTT GTT ATA CCT ATA GGC ACA T CLU5-mRNA-Probe, (SEQ ID NO: 36)FAM-5′-GCC ATT CCA CTC AGC AGA ACG TCT G-TAMRA Tilapia beta-actinTIL-Actin-qF1, (SEQ ID NO: 37) 5′-GCG TGA CAT CAA RGA GAA GCT GTIL-Actin-qR1, (SEQ ID NO: 38) 5′-CCA ATG GTG ATG ACC TGT CTIL-Actin-Probe, (SEQ ID NO: 39)FAM-5′-CCC TGG AGA AGA GTT ACG AGC TGC-TAMRA

As shown in FIG. 7C, detection of authentic TiLV was found in varioustissue samples of diseased tilapia from Israel using real-time PCR withprimers specific for TiLV.

REFERENCES

-   Agius and Roberts 2003. J. Fish Dis. 26:499-509.-   Altschul et al. 1997. Nucleic Acids Res. 25:3389-3402.-   Bacharach et al. 2000. J. Virol. 74:11027-11039.-   Bigarré et al. 2010. J. Fish Dis. 32:667-673.-   Brawand et al. 2014. Nature 513:375-81.-   Camacho et al. 2009. BMC Bioinformatics 10:421.-   Crandell et al. 1975. J. Clin. Microbiol. 2:465-468.-   Food and Agriculture Organization of the United Nations (FAO). 2010.    Cultured aquatic species information programme, Oreochromis    niloticus (Linnaeus, 1758). Food and Agriculture Organization of the    United Nations, Rome, Italy.-   Food and Agriculture Organization of the United Nations (FAO). 2010.    Fisheries and Aquaculture Department. Species fact sheets:    Oreochromis niloticus (Linnaeus, 1758).-   Food and Agriculture Organization of the United Nations, Rome,    Italy.-   Hasegawa et al. 1997. Fish Pathol. 32:127-128.-   Hedrick et al. 2000. J. Aquat. Anim. Health 12:44-57.-   Hutoran et al. 2005. J. Viral. 79:1983-1991.-   Johnson et al. 2008. Nucleic Acids Res. 36(Suppl 2):W5-W9.-   Laham and Bacharach 2007 J. Virol. 81:10687-10698.-   Li 2013. arXiv:1303.3997.-   Martin 2011 EMBnet.journal 17:10-12.-   Melamed et al. 2004. J. Virol. 78:9675-9688.-   Nehls and Boehm 1995. Trends Genet. 11:39-40.-   Oberpichler et al. 2008. Environ. Microbiol. 10:2020-2029.-   Pruitt et al. 2012 Nucleic Acids Res. 40:D130-D135.-   Reed and Mucnch 1938. Am. J. Hyg. 27:493-497.-   Reynolds 1963. J. Cell. Biol. 17:208-212.-   Roberts et al. 2012 Laboratory Methods in Fish Pathology, Fourth    Edition, John Wiley & Sons, Inc.-   Scotto-Lavino et al. 2006a. Nat. Protoc. 1:2742-2745.-   Scotto-Lavino et al. 2006b. Nat. Protoc. 1:2555-2562.-   Shlapobersky et al. 2010. Virology 399:239-247.-   Yang et al. 2012. BMC Genomics 13:475.

1.-12. (canceled)
 13. An isolated polypeptide encoded by the nucleicacid having a sequence selected from the group consisting of SEQ ID NOs:1-11 or a nucleic acid complementary to a sequence selected from thegroup consisting of SEQ ID NOs: 1-11.
 14. (canceled)
 15. An isolatedpolypeptide encoded by a variant of any of one of SEQ ID NOs: 1-11 or avariant of a nucleic acid complementary to a sequence selected from thegroup consisting of SEQ ID NOs; 1-11, wherein said variant has at leastabout 80% sequence identity to SEQ ID NOs: 1-11 or a nucleic acidcomplementary to the sequence of any of SEQ ID NOs: 1-11.
 16. Theisolated polypeptide of claim 15, wherein the variant has at least about85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%,or about 99.5% identity to any of SEQ ID NOs: 1-11 or a sequencecomplementary to any of SEQ ID NOs: 1-11.
 17. (canceled)
 18. (canceled)19. An isolated polypeptide comprising the amino acid sequence of SEQ IDNO: 12 or comprising an amino acid sequence that has at least about 80%sequence identity to SEQ ID NO:
 12. 20.-22. (canceled)
 23. An isolatedantibody that specifically binds to a polypeptide encoded by the nucleicacid having a sequence selected from the group consisting of SEQ ID NOs:1-11, fragments and variants thereof or a polypeptide comprising theamino acid sequence of SEQ ID NO: 12, fragments and variants thereof.24. (canceled)
 25. (canceled)
 26. The isolated antibody of claim 23,wherein the antibody binds to Tilapia Lake Virus or a Tilapia Lake Viruspolypeptide and inhibits, neutralizes or reduces the function oractivity of the Tilapia Lake Virus or the Tilapia Lake Viruspolypeptide. 27.-35. (canceled)
 36. An immunogenic compositioncomprising a Tilapia Lake Virus polypeptide, according to claim
 13. 37.(canceled)
 38. (canceled)
 39. (canceled)
 40. An immunogenic compositioncomprising a Tilapia Lake Virus polypeptide, according to claim
 15. 41.An immunogenic composition comprising a Tilapia Lake Virus polypeptide,according to claim
 16. 42. An immunogenic composition comprising aTilapia Lake Virus polypeptide, according to claim
 19. 43. (canceled)44. (canceled)
 45. (canceled)
 46. An immunogenic composition comprisinga killed virus and/or attenuated virus comprising a Tilapia Lake Viruspolypeptide.
 47. (canceled)
 48. The immunogenic composition of claim 46further comprising at least one excipient, additive or adjuvant. 49.(canceled)
 50. (canceled)
 51. A method of inducing an immune response inan animal, the method comprising administering the immunogeniccomposition of claim
 40. 52. A method for preventing or reducing aTilapia Lake Viral infection in an animal, the method comprisingadministering the immunogenic composition of claim
 40. 53. The method ofclaim 51, wherein the immunogenic composition is administered orally, byimmersion or by injection. 54.-65. (canceled)
 66. A method fordetermining whether or not a sample contains Tilapia Lake Virus or hasbeen infected by Tilapia Lake Virus, the method comprising: a)contacting a biological sample with the antibody of claim 23; and b)determining whether or not the antibody binds to an antigen in thebiological sample, wherein binding indicates that the biological samplecontains Tilapia Lake Virus. 67.-81. (canceled)
 82. A method of inducingan immune response in an animal, the method comprising administering theimmunogenic composition of claim
 41. 83. A method for preventing orreducing a Tilapia Lake Viral infection in an animal, the methodcomprising administering the immunogenic composition of claim
 41. 84.The method of claim 83, wherein the immunogenic composition isadministered orally, by immersion or by injection.
 85. A method ofinducing an immune response in an animal, the method comprisingadministering the immunogenic composition of claim
 42. 86. A method forpreventing or reducing a Tilapia Lake Viral infection in an animal, themethod comprising administering the immunogenic composition of claim 42.87. The method of claim 86, wherein the immunogenic composition isadministered orally, by immersion or by injection.